Structured Review

Thermo Fisher rna
Agarose gel electrophoresis of <t>RDA</t> products ( A ) and its hybridized autoradiogram ( B ). In vitro transcribed <t>RNA</t> and total cellular RNA were mixed as described in the legend to Figure 2 . Double-stranded cDNAs were synthesized and subjected to RDA as described in Materials and Methods. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide ( A ), blotted onto a nylon membrane and hybridized with 32 P-labelled pCIneo ( B ). Positions and sizes (bp) of markers are present on the left.
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Images

1) Product Images from "Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription"

Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

Journal: Nucleic Acids Research

doi: 10.1093/nar/gni064

Agarose gel electrophoresis of RDA products ( A ) and its hybridized autoradiogram ( B ). In vitro transcribed RNA and total cellular RNA were mixed as described in the legend to Figure 2 . Double-stranded cDNAs were synthesized and subjected to RDA as described in Materials and Methods. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide ( A ), blotted onto a nylon membrane and hybridized with 32 P-labelled pCIneo ( B ). Positions and sizes (bp) of markers are present on the left.
Figure Legend Snippet: Agarose gel electrophoresis of RDA products ( A ) and its hybridized autoradiogram ( B ). In vitro transcribed RNA and total cellular RNA were mixed as described in the legend to Figure 2 . Double-stranded cDNAs were synthesized and subjected to RDA as described in Materials and Methods. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide ( A ), blotted onto a nylon membrane and hybridized with 32 P-labelled pCIneo ( B ). Positions and sizes (bp) of markers are present on the left.

Techniques Used: Agarose Gel Electrophoresis, In Vitro, Synthesized, Amplification, Staining

Agarose gel electrophoresis of RDA products from RNA extracted from bovine parainfluenza virus 3-infected cells. Double-stranded cDNA was synthesized from RNA of bovine parainfluenza virus 3-infected MDBK cells and subjected to RDA. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide. RDA product from the uninfected control cells was used as a negative control.
Figure Legend Snippet: Agarose gel electrophoresis of RDA products from RNA extracted from bovine parainfluenza virus 3-infected cells. Double-stranded cDNA was synthesized from RNA of bovine parainfluenza virus 3-infected MDBK cells and subjected to RDA. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide. RDA product from the uninfected control cells was used as a negative control.

Techniques Used: Agarose Gel Electrophoresis, Infection, Synthesized, Amplification, Staining, Negative Control

Agarose gel electrophoresis of RDA products with PCR products used for probes for hybridization ( A ) and a hybridized fluorogram ( B ). RNA was extracted from SARS-CoV-infected cells and subjected to RDA according to the method described in Materials and Methods. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gels and blotted on a Nylon membrane. The membrane was then cut into slits that contained the lane showing the presence of DNA. On the other hand, the PCR fragments predicted to be amplified in the RDA reaction were amplified and subsequently ascertained by agarose gel electrophoresis (A). The amplified genomic fragments of SARS-CoV were Dig-labelled and used as probes for hybridization to each slit of the Nylon membrane containing the RDA product. Hybridization was performed in separate hybridization bags. After washing with 1× SSC and 0.1% SDS solution, the hybridized probes were detected on a fluorogram (B). Positions and sizes (bp) of markers are present on the left.
Figure Legend Snippet: Agarose gel electrophoresis of RDA products with PCR products used for probes for hybridization ( A ) and a hybridized fluorogram ( B ). RNA was extracted from SARS-CoV-infected cells and subjected to RDA according to the method described in Materials and Methods. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gels and blotted on a Nylon membrane. The membrane was then cut into slits that contained the lane showing the presence of DNA. On the other hand, the PCR fragments predicted to be amplified in the RDA reaction were amplified and subsequently ascertained by agarose gel electrophoresis (A). The amplified genomic fragments of SARS-CoV were Dig-labelled and used as probes for hybridization to each slit of the Nylon membrane containing the RDA product. Hybridization was performed in separate hybridization bags. After washing with 1× SSC and 0.1% SDS solution, the hybridized probes were detected on a fluorogram (B). Positions and sizes (bp) of markers are present on the left.

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Hybridization, Infection, Amplification

Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.
Figure Legend Snippet: Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.

Techniques Used: Synthesized, In Vitro, Plasmid Preparation, Agarose Gel Electrophoresis

2) Product Images from "Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription"

Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

Journal: Nucleic Acids Research

doi: 10.1093/nar/gni064

Agarose gel electrophoresis of RDA products from RNA extracted from bovine parainfluenza virus 3-infected cells. Double-stranded cDNA was synthesized from RNA of bovine parainfluenza virus 3-infected MDBK cells and subjected to RDA. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide. RDA product from the uninfected control cells was used as a negative control.
Figure Legend Snippet: Agarose gel electrophoresis of RDA products from RNA extracted from bovine parainfluenza virus 3-infected cells. Double-stranded cDNA was synthesized from RNA of bovine parainfluenza virus 3-infected MDBK cells and subjected to RDA. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide. RDA product from the uninfected control cells was used as a negative control.

Techniques Used: Agarose Gel Electrophoresis, Infection, Synthesized, Amplification, Staining, Negative Control

3) Product Images from "A PIWI homolog is one of the proteins expressed exclusively during macronuclear development in the ciliate Stylonychia lemnae"

Article Title: A PIWI homolog is one of the proteins expressed exclusively during macronuclear development in the ciliate Stylonychia lemnae

Journal: Nucleic Acids Research

doi:

Detection of stage-specific RNAs and the corresponding macronuclear genes. Total RNA of vegetative cells and different stages during macronuclear development was isolated, first and second strand cDNA synthesis was performed (see Materials and Methods). For agarose gel electrophoresis equal volumes from the same cDNA preparation were used for each of the four gels. Lane 1, cDNA derived from vegetative cells; lanes 2–5, cDNA derived from exconjugants, 0, 20, 30 or 40 h PC, respectively. The gels were blotted and hybridized with Dig labeled probes generated by PCR amplification of the differentially expressed clones mdp1 ( A ), mdp2 ( B ) and mdp3 ( C ). To check the integrity of the different cDNAs, actin I was used as a control ( D ). Macronuclear DNA was isolated and separated by agarose gel electrophoresis. The gels were blotted and hybridized with the corresponding Dig labeled probes (lane 6).
Figure Legend Snippet: Detection of stage-specific RNAs and the corresponding macronuclear genes. Total RNA of vegetative cells and different stages during macronuclear development was isolated, first and second strand cDNA synthesis was performed (see Materials and Methods). For agarose gel electrophoresis equal volumes from the same cDNA preparation were used for each of the four gels. Lane 1, cDNA derived from vegetative cells; lanes 2–5, cDNA derived from exconjugants, 0, 20, 30 or 40 h PC, respectively. The gels were blotted and hybridized with Dig labeled probes generated by PCR amplification of the differentially expressed clones mdp1 ( A ), mdp2 ( B ) and mdp3 ( C ). To check the integrity of the different cDNAs, actin I was used as a control ( D ). Macronuclear DNA was isolated and separated by agarose gel electrophoresis. The gels were blotted and hybridized with the corresponding Dig labeled probes (lane 6).

Techniques Used: Isolation, Agarose Gel Electrophoresis, Derivative Assay, Labeling, Generated, Polymerase Chain Reaction, Amplification, Clone Assay

4) Product Images from "Diffractometric Detection of Proteins using Microbead-based Rolling Circle Amplification"

Article Title: Diffractometric Detection of Proteins using Microbead-based Rolling Circle Amplification

Journal: Analytical chemistry

doi: 10.1021/ac901716d

(a) Schematic of microbead-based aptamer sandwich assay without RCA. Biotinylated aptamer was immobilized on streptavidin functionalized periodic patterns. PDGF-BB was introduced, and sandwiched by another biotinylated aptamer. Streptavidin conjugated
Figure Legend Snippet: (a) Schematic of microbead-based aptamer sandwich assay without RCA. Biotinylated aptamer was immobilized on streptavidin functionalized periodic patterns. PDGF-BB was introduced, and sandwiched by another biotinylated aptamer. Streptavidin conjugated

Techniques Used:

(a)-(e) Optical micrographs of the self-assembled diffraction gratings formed by streptavidin-labeled beads with varying PDGF-BB concentration (10 pM – 100 nM). (f) Grating with no PDGF-BB.
Figure Legend Snippet: (a)-(e) Optical micrographs of the self-assembled diffraction gratings formed by streptavidin-labeled beads with varying PDGF-BB concentration (10 pM – 100 nM). (f) Grating with no PDGF-BB.

Techniques Used: Labeling, Concentration Assay

(a) Schematic of RCA-based microbead detection assay in combination with aptamers. A biotinylated anti-PDGF-B specific aptamer is immobilized on streptavidin coated periodic patterns. PDGF-BB is introduced and captured by the aptamer. An aptamer-primer
Figure Legend Snippet: (a) Schematic of RCA-based microbead detection assay in combination with aptamers. A biotinylated anti-PDGF-B specific aptamer is immobilized on streptavidin coated periodic patterns. PDGF-BB is introduced and captured by the aptamer. An aptamer-primer

Techniques Used: Detection Assay

5) Product Images from "Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription"

Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

Journal: Nucleic Acids Research

doi: 10.1093/nar/gni064

Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.
Figure Legend Snippet: Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.

Techniques Used: Synthesized, In Vitro, Plasmid Preparation, Agarose Gel Electrophoresis

6) Product Images from "Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *"

Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.384321

SDS-PAGE analysis of purified DNA polymerase I, DNA ligase I, and the Vsr endonuclease. Purified proteins (3 μg) were resolved on an 11% polyacrylamide gel run in the presence of SDS and stained with Coomassie Blue. Lane 1 , molecular mass standards
Figure Legend Snippet: SDS-PAGE analysis of purified DNA polymerase I, DNA ligase I, and the Vsr endonuclease. Purified proteins (3 μg) were resolved on an 11% polyacrylamide gel run in the presence of SDS and stained with Coomassie Blue. Lane 1 , molecular mass standards

Techniques Used: SDS Page, Purification, Staining

DNA polymerase I and the Vsr endonuclease are sufficient to repair a T:G mismatch. The VSP repair reaction contained 50 ng of covalently closed heteroduplex substrate, 52 n m Vsr endonuclease, and a titration of DNA polymerase I from 467 to 0.2 n m as described
Figure Legend Snippet: DNA polymerase I and the Vsr endonuclease are sufficient to repair a T:G mismatch. The VSP repair reaction contained 50 ng of covalently closed heteroduplex substrate, 52 n m Vsr endonuclease, and a titration of DNA polymerase I from 467 to 0.2 n m as described

Techniques Used: Titration

DNA ligase I seals the nick created by DNA polymerase I nick translation. Repair and ligation reactions were conducted as described under “Materials and Methods” using 200 ng of pUC19-VSR heteroduplex DNA (∼1.2 n m molecules), 10
Figure Legend Snippet: DNA ligase I seals the nick created by DNA polymerase I nick translation. Repair and ligation reactions were conducted as described under “Materials and Methods” using 200 ng of pUC19-VSR heteroduplex DNA (∼1.2 n m molecules), 10

Techniques Used: Nick Translation, Ligation

Vsr-catalyzed nicking of a covalently closed circular DNA substrate. Vsr-dependent nicking reactions were as described under “Materials and Methods.” A , the titration of the Vsr endonuclease was from 153 to 0 n m with each DNA substrate,
Figure Legend Snippet: Vsr-catalyzed nicking of a covalently closed circular DNA substrate. Vsr-dependent nicking reactions were as described under “Materials and Methods.” A , the titration of the Vsr endonuclease was from 153 to 0 n m with each DNA substrate,

Techniques Used: Titration

The impact of DNA ligase I concentration on repair track length. Ligation and repair reactions were as described under “Materials and Methods” using 200 ng of covalently closed pUC19-VSR heteroduplex DNA (∼1.2 n m molecules), 10
Figure Legend Snippet: The impact of DNA ligase I concentration on repair track length. Ligation and repair reactions were as described under “Materials and Methods” using 200 ng of covalently closed pUC19-VSR heteroduplex DNA (∼1.2 n m molecules), 10

Techniques Used: Concentration Assay, Ligation

The Vsr endonuclease incises DNA immediately 5′ to the mismatched thymidine. Vsr nicking reactions were as described under “Materials and Methods.” The Vsr endonuclease was incubated with covalently closed pUC19-VSR heteroduplex
Figure Legend Snippet: The Vsr endonuclease incises DNA immediately 5′ to the mismatched thymidine. Vsr nicking reactions were as described under “Materials and Methods.” The Vsr endonuclease was incubated with covalently closed pUC19-VSR heteroduplex

Techniques Used: Incubation

7) Product Images from "Human COX7A2L Regulates Complex III Biogenesis and Promotes Supercomplex Organization Remodeling without Affecting Mitochondrial Bioenergetics"

Article Title: Human COX7A2L Regulates Complex III Biogenesis and Promotes Supercomplex Organization Remodeling without Affecting Mitochondrial Bioenergetics

Journal: Cell reports

doi: 10.1016/j.celrep.2018.10.058

The Short-COX7A2L Variant Binds to CIII2 but Does Not Rescue SC III2+IV Assembly in COX7A2L-KO Cells Control HEK293T cells (WT) and both COX7A2L - KO (KO1 and KO2) clones carrying an empty vector (ev) or constructs to overexpress COX7A2L-Myc- DDK (long) or short-COX7A2L-Myc-DDK (short) were used in the following experiments. (A) SDS-PAGE followed by immunoblotting to estimate steady-state levels of endogenous COX7A2L (~12.6 kDa) from exogenous COX7A2L-Myc-DDK (~16.2 kDa). Membranes were also incubated with antibodies that recognize the indicated OXPHOS subunits. (B). 2D-BN/SDS-PAGE and immunoblotting using digitonin-solubilized mitochondrial extracts (detergent/protein ratio, 4:1) and the indicated antibodies. (C and D) BN-PAGE followed by immunoblotting in digitonized whole-cell extracts (C) or by CI-IGA and CIV-IGA assays and/or immunoblotting in digitonized isolated mitochondria (D). Membranes were incubated with the indicated antibodies. I+III 2 +IV n , SCs containing CI, CIII 2 , and CIV. I+III 2 , SC containing CI and CIII 2 . III 2 +IV, SC containing CIII 2 and CIV. III 2 , complex III dimer (CIII 2 ). IV, complex IV; IV 2 , complex IV dimer (CIV 2 ); II, complex II. Subcomplexes that contain COX1 are indicated as subCOX1. In (D), an unidentified band running a bit faster than the SC III 2 +IV cross-reacting with the COX5B antibody (Ab) (but not with the CORE2 Ab) is indicated with an asterisk.
Figure Legend Snippet: The Short-COX7A2L Variant Binds to CIII2 but Does Not Rescue SC III2+IV Assembly in COX7A2L-KO Cells Control HEK293T cells (WT) and both COX7A2L - KO (KO1 and KO2) clones carrying an empty vector (ev) or constructs to overexpress COX7A2L-Myc- DDK (long) or short-COX7A2L-Myc-DDK (short) were used in the following experiments. (A) SDS-PAGE followed by immunoblotting to estimate steady-state levels of endogenous COX7A2L (~12.6 kDa) from exogenous COX7A2L-Myc-DDK (~16.2 kDa). Membranes were also incubated with antibodies that recognize the indicated OXPHOS subunits. (B). 2D-BN/SDS-PAGE and immunoblotting using digitonin-solubilized mitochondrial extracts (detergent/protein ratio, 4:1) and the indicated antibodies. (C and D) BN-PAGE followed by immunoblotting in digitonized whole-cell extracts (C) or by CI-IGA and CIV-IGA assays and/or immunoblotting in digitonized isolated mitochondria (D). Membranes were incubated with the indicated antibodies. I+III 2 +IV n , SCs containing CI, CIII 2 , and CIV. I+III 2 , SC containing CI and CIII 2 . III 2 +IV, SC containing CIII 2 and CIV. III 2 , complex III dimer (CIII 2 ). IV, complex IV; IV 2 , complex IV dimer (CIV 2 ); II, complex II. Subcomplexes that contain COX1 are indicated as subCOX1. In (D), an unidentified band running a bit faster than the SC III 2 +IV cross-reacting with the COX5B antibody (Ab) (but not with the CORE2 Ab) is indicated with an asterisk.

Techniques Used: Variant Assay, Clone Assay, Plasmid Preparation, Construct, SDS Page, Incubation, Polyacrylamide Gel Electrophoresis, Isolation

8) Product Images from "Long non-coding RNA regulation of spermatogenesis and endosomal processes via the spectrin cytoskeleton in Drosophila"

Article Title: Long non-coding RNA regulation of spermatogenesis and endosomal processes via the spectrin cytoskeleton in Drosophila

Journal: bioRxiv

doi: 10.1101/2020.07.24.220640

The lncRNA CR45362 biochemically interacts with cytoskeletal and endosomal related proteins (A) Map of ChIRP probe locations relative to CR45362 gene. Probes 5-8 map to the deleted segment of the gene in the KO. (Image: SnapGene 4.3.4) (B) Molecular Function Gene Ontology for top ChIRP-MS proteins indicates cytoskeletal proteins bind CR45362. (C) Driver test indicates Ptc-Gal4 x UAS-GFP expresses GFP in the same region of the basal testis as is indicated by CR45362 FISH (see also Fig. 2A). (D) RNAi screen of top ChIRP-MS candidate proteins. Five pairs of testis from 3-5 day old males from each UAS-Gal4 cross were dissected of which we scored 3. If defects were seen, we repeated this screening for a second round of crosses. If both testis of a pair had the same phenotype, they were scored according to the following criteria: normal nuclear bundling, one or more scattered nuclear bundles, no nuclear bundles present, one or more individualization complexes (actin) scattered, no actin cones present, or testis deformed. (E) Results from ChIRP-MS. Columns list the number of hits (spectra) for each protein. Numbers are internally relative to each individual sample, and are used for ranking proteins, cut-off selection, and identifying background proteins. Probes 5-8 (Fig. 4A, S3) cover the CRISPR excised region, when mixed with KO samples these probes serve to identify background proteins pulled down by beads, general biotin probes, and as a negative control for the pulldown of CR45362 (Fig. S4). Each individual probe could have probe-specific background, therefore established protocols54 recommend the optimal control for a ChIRP assay is to pool probes into “Even or Odd” groups in WT samples, allowing further identification of background proteins and can be compared with the WT sample mixed with “All” probes. (F) The Ptc-Gal4 x α-Spec RNAi line has dispersed actin cones (indicated by phalloidin staining) and spermatid nuclei (yellow arrows), which phenocopied the nuclear dispersion seen in CR45362KO (G). *All scale bars represent 10 μm.
Figure Legend Snippet: The lncRNA CR45362 biochemically interacts with cytoskeletal and endosomal related proteins (A) Map of ChIRP probe locations relative to CR45362 gene. Probes 5-8 map to the deleted segment of the gene in the KO. (Image: SnapGene 4.3.4) (B) Molecular Function Gene Ontology for top ChIRP-MS proteins indicates cytoskeletal proteins bind CR45362. (C) Driver test indicates Ptc-Gal4 x UAS-GFP expresses GFP in the same region of the basal testis as is indicated by CR45362 FISH (see also Fig. 2A). (D) RNAi screen of top ChIRP-MS candidate proteins. Five pairs of testis from 3-5 day old males from each UAS-Gal4 cross were dissected of which we scored 3. If defects were seen, we repeated this screening for a second round of crosses. If both testis of a pair had the same phenotype, they were scored according to the following criteria: normal nuclear bundling, one or more scattered nuclear bundles, no nuclear bundles present, one or more individualization complexes (actin) scattered, no actin cones present, or testis deformed. (E) Results from ChIRP-MS. Columns list the number of hits (spectra) for each protein. Numbers are internally relative to each individual sample, and are used for ranking proteins, cut-off selection, and identifying background proteins. Probes 5-8 (Fig. 4A, S3) cover the CRISPR excised region, when mixed with KO samples these probes serve to identify background proteins pulled down by beads, general biotin probes, and as a negative control for the pulldown of CR45362 (Fig. S4). Each individual probe could have probe-specific background, therefore established protocols54 recommend the optimal control for a ChIRP assay is to pool probes into “Even or Odd” groups in WT samples, allowing further identification of background proteins and can be compared with the WT sample mixed with “All” probes. (F) The Ptc-Gal4 x α-Spec RNAi line has dispersed actin cones (indicated by phalloidin staining) and spermatid nuclei (yellow arrows), which phenocopied the nuclear dispersion seen in CR45362KO (G). *All scale bars represent 10 μm.

Techniques Used: Fluorescence In Situ Hybridization, Selection, CRISPR, Negative Control, Staining

The lncRNA CR45362 has positive effect on endo/lysosomal activity and fertility A) Diagram of CR45362, SNP’s identified in our lysosomal GWAS, primers, and CRISPR gRNAs. (Image: SnapGene 4.3.4) (B) DNA gel using primers flanking deletion target segment (p473 474). WT has band at 2182 demonstrating the primer targeted segment has no deletion, heterozygous CR45362 KO (+/-) has the 2182bp band but also the expected 508bp band for the deletion on the homologous chromosome, and KO has only the 508bp band indicating successful deletion in the CR45362 gene. The 508bp band was excised for sequencing and alignment (Fig. S1). (C) DNA gel of cDNA converted from CR45362 RNA using primers 1421 and 1423 (as diagrammed in 1A) that precisely correspond to the predicted ends of the lncRNA. The predicted size of CR45362 if spliced as annotated is 1207bp while unspliced is 1338bp. The gel revealed only a single band near the 1200bp mark indicating that there is a single isoform in our WT line and it is consistent with predicted splicing. Full length gel can be found in Fig. S1. (D,E) Lysotracker staining of WT and CR45362KO fed and fasted fat bodies. Knockout of the lncRNA CR45362 results in lowered Lysotracker staining under both conditions. One replicate consists of the average of three circular areas of 100μm per fly fat body measured by Cell Sens software for percentage of measured area containing fluorescence. From left to right n=7,12,6,7,6,8. Fed/fasting interaction calculated using Two-way Anova column factor (** p = 0.0034), WT vs KO fed two tailed unpaired t-test (*** p = 0.001) mean+/-s.e.m. (F) Fertility was tested by crossing individual CR45362KO male flies with WT, KO, or heterozygotes for the KO virgin females. These crosses were unable to produce offspring. However, KO and heterozygous females produced adult progeny and were fully fertile, indicating a male homozygous KO sterility defect. From top to bottom n=4,13,27,3,2,5,3,6. *All scale bars represent 10 μm. *For all figures: WT = (w1118), +/-= Heterozygous, KO = CR45362 mutant.
Figure Legend Snippet: The lncRNA CR45362 has positive effect on endo/lysosomal activity and fertility A) Diagram of CR45362, SNP’s identified in our lysosomal GWAS, primers, and CRISPR gRNAs. (Image: SnapGene 4.3.4) (B) DNA gel using primers flanking deletion target segment (p473 474). WT has band at 2182 demonstrating the primer targeted segment has no deletion, heterozygous CR45362 KO (+/-) has the 2182bp band but also the expected 508bp band for the deletion on the homologous chromosome, and KO has only the 508bp band indicating successful deletion in the CR45362 gene. The 508bp band was excised for sequencing and alignment (Fig. S1). (C) DNA gel of cDNA converted from CR45362 RNA using primers 1421 and 1423 (as diagrammed in 1A) that precisely correspond to the predicted ends of the lncRNA. The predicted size of CR45362 if spliced as annotated is 1207bp while unspliced is 1338bp. The gel revealed only a single band near the 1200bp mark indicating that there is a single isoform in our WT line and it is consistent with predicted splicing. Full length gel can be found in Fig. S1. (D,E) Lysotracker staining of WT and CR45362KO fed and fasted fat bodies. Knockout of the lncRNA CR45362 results in lowered Lysotracker staining under both conditions. One replicate consists of the average of three circular areas of 100μm per fly fat body measured by Cell Sens software for percentage of measured area containing fluorescence. From left to right n=7,12,6,7,6,8. Fed/fasting interaction calculated using Two-way Anova column factor (** p = 0.0034), WT vs KO fed two tailed unpaired t-test (*** p = 0.001) mean+/-s.e.m. (F) Fertility was tested by crossing individual CR45362KO male flies with WT, KO, or heterozygotes for the KO virgin females. These crosses were unable to produce offspring. However, KO and heterozygous females produced adult progeny and were fully fertile, indicating a male homozygous KO sterility defect. From top to bottom n=4,13,27,3,2,5,3,6. *All scale bars represent 10 μm. *For all figures: WT = (w1118), +/-= Heterozygous, KO = CR45362 mutant.

Techniques Used: Activity Assay, GWAS, CRISPR, Sequencing, Staining, Knock-Out, Software, Fluorescence, Two Tailed Test, Produced, Sterility, Mutagenesis

9) Product Images from "Genetic polymorphisms in bone morphogenetic protein receptor type IA gene predisposes individuals to ossification of the posterior longitudinal ligament of the cervical spine via the smad signaling pathway"

Article Title: Genetic polymorphisms in bone morphogenetic protein receptor type IA gene predisposes individuals to ossification of the posterior longitudinal ligament of the cervical spine via the smad signaling pathway

Journal: BMC Musculoskeletal Disorders

doi: 10.1186/s12891-018-1966-1

The BMPR-IA gene coding sequence and pGEM-T/BMPR-IA (WT) vector digested with Hind III/Bam HI enzyme were analyzed by 4% agarose gel electrophoresis, respectively. a The annealed ds-oligos of BMPR-IA gene coding sequence was analyzed by 4% agarose gel electrophoresis. Each ds oligo annealing reactant showed a detectable molecular weight band around 1599 bp, as expected for the length of the designed ds-oligos of BMPR-IA gene. Lane M: Marker, Lane B: BMPR-IA gene.0020 b After the pGEM-T/BMPR-IA (WT) vector had been digested with Hind III/Bam HI enzyme, the fragments for BMPR-IA cDNA (1599 bp) and the pGEM-T vector (3000 bp) were observed in all the PCR products as analyzed by 4% agarose gel electrophoresis, respectively. The results showed that the ds-oligos of the BMPR-IA gene cDNA were ligated into the pGEM-T vector. Lane M: Marker, Lane 1: pGEM-T/BMPR-IA (WT) vector, Lane 2: The pGEM-T vector and BMPR-IA cDNA
Figure Legend Snippet: The BMPR-IA gene coding sequence and pGEM-T/BMPR-IA (WT) vector digested with Hind III/Bam HI enzyme were analyzed by 4% agarose gel electrophoresis, respectively. a The annealed ds-oligos of BMPR-IA gene coding sequence was analyzed by 4% agarose gel electrophoresis. Each ds oligo annealing reactant showed a detectable molecular weight band around 1599 bp, as expected for the length of the designed ds-oligos of BMPR-IA gene. Lane M: Marker, Lane B: BMPR-IA gene.0020 b After the pGEM-T/BMPR-IA (WT) vector had been digested with Hind III/Bam HI enzyme, the fragments for BMPR-IA cDNA (1599 bp) and the pGEM-T vector (3000 bp) were observed in all the PCR products as analyzed by 4% agarose gel electrophoresis, respectively. The results showed that the ds-oligos of the BMPR-IA gene cDNA were ligated into the pGEM-T vector. Lane M: Marker, Lane 1: pGEM-T/BMPR-IA (WT) vector, Lane 2: The pGEM-T vector and BMPR-IA cDNA

Techniques Used: IA, Sequencing, Plasmid Preparation, Agarose Gel Electrophoresis, Molecular Weight, Marker, Polymerase Chain Reaction

10) Product Images from "A PIWI homolog is one of the proteins expressed exclusively during macronuclear development in the ciliate Stylonychia lemnae"

Article Title: A PIWI homolog is one of the proteins expressed exclusively during macronuclear development in the ciliate Stylonychia lemnae

Journal: Nucleic Acids Research

doi:

Detection of stage-specific RNAs and the corresponding macronuclear genes. Total RNA of vegetative cells and different stages during macronuclear development was isolated, first and second strand cDNA synthesis was performed (see Materials and Methods). For agarose gel electrophoresis equal volumes from the same cDNA preparation were used for each of the four gels. Lane 1, cDNA derived from vegetative cells; lanes 2–5, cDNA derived from exconjugants, 0, 20, 30 or 40 h PC, respectively. The gels were blotted and hybridized with Dig labeled probes generated by PCR amplification of the differentially expressed clones mdp1 ( A ), mdp2 ( B ) and mdp3 ( C ). To check the integrity of the different cDNAs, actin I was used as a control ( D ). Macronuclear DNA was isolated and separated by agarose gel electrophoresis. The gels were blotted and hybridized with the corresponding Dig labeled probes (lane 6).
Figure Legend Snippet: Detection of stage-specific RNAs and the corresponding macronuclear genes. Total RNA of vegetative cells and different stages during macronuclear development was isolated, first and second strand cDNA synthesis was performed (see Materials and Methods). For agarose gel electrophoresis equal volumes from the same cDNA preparation were used for each of the four gels. Lane 1, cDNA derived from vegetative cells; lanes 2–5, cDNA derived from exconjugants, 0, 20, 30 or 40 h PC, respectively. The gels were blotted and hybridized with Dig labeled probes generated by PCR amplification of the differentially expressed clones mdp1 ( A ), mdp2 ( B ) and mdp3 ( C ). To check the integrity of the different cDNAs, actin I was used as a control ( D ). Macronuclear DNA was isolated and separated by agarose gel electrophoresis. The gels were blotted and hybridized with the corresponding Dig labeled probes (lane 6).

Techniques Used: Isolation, Agarose Gel Electrophoresis, Derivative Assay, Labeling, Generated, Polymerase Chain Reaction, Amplification, Clone Assay

11) Product Images from "Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes"

Article Title: Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0107351

Full-length enrichment for library cloning and next generation sequencing (NGS). Full-length (blue line with 5′ cap) or truncated (short blue line without 5′ cap) mRNAs were reverse transcribed into first-strand cDNA using oligo-dT primers (red arrow). The mRNA:cDNA hybrid was treated with RNase I (scissor) to remove the single-stranded RNA that was not fully extended by the first-strand cDNA, followed by selection for full-length transcripts using Cap-antibody magnetic beads to enrich the full-length mRNA:cDNA. The full-length single-stranded DNA (FLssDNA) was eluted from beads and used for both cDNA library cloning (lower left) and NGS (lower right). For full-length library cloning, a double-stranded adaptor (green) was linked to the 5′ end of ssDNA. Second-strand cDNA synthesis was then carried out, followed by cloning into a vector. For NGS, the full-length enriched ssDNA was fragmented by sonication to target fragments in the range of 200–400 bp, followed by ligation of the double-stranded DNA sequencing adaptor mixture (purple) to 3′ and 5′ ends of ssDNA. To maintain the complexity of the library while enriching the full-length cDNA for NGS, the original polyA mRNA was also fragmented using RNAse III, followed by ligation of the double-stranded RNA sequencing adaptor mixture (brown) to 3′ and 5′ ends of mRNA. After first- and second-strand synthesis, the polyA and capped mRNA and polyA and non-capped mRNA samples were mixed in a 3∶1 ratio and applied to the downstream NGS procedure.
Figure Legend Snippet: Full-length enrichment for library cloning and next generation sequencing (NGS). Full-length (blue line with 5′ cap) or truncated (short blue line without 5′ cap) mRNAs were reverse transcribed into first-strand cDNA using oligo-dT primers (red arrow). The mRNA:cDNA hybrid was treated with RNase I (scissor) to remove the single-stranded RNA that was not fully extended by the first-strand cDNA, followed by selection for full-length transcripts using Cap-antibody magnetic beads to enrich the full-length mRNA:cDNA. The full-length single-stranded DNA (FLssDNA) was eluted from beads and used for both cDNA library cloning (lower left) and NGS (lower right). For full-length library cloning, a double-stranded adaptor (green) was linked to the 5′ end of ssDNA. Second-strand cDNA synthesis was then carried out, followed by cloning into a vector. For NGS, the full-length enriched ssDNA was fragmented by sonication to target fragments in the range of 200–400 bp, followed by ligation of the double-stranded DNA sequencing adaptor mixture (purple) to 3′ and 5′ ends of ssDNA. To maintain the complexity of the library while enriching the full-length cDNA for NGS, the original polyA mRNA was also fragmented using RNAse III, followed by ligation of the double-stranded RNA sequencing adaptor mixture (brown) to 3′ and 5′ ends of mRNA. After first- and second-strand synthesis, the polyA and capped mRNA and polyA and non-capped mRNA samples were mixed in a 3∶1 ratio and applied to the downstream NGS procedure.

Techniques Used: Clone Assay, Next-Generation Sequencing, Selection, Magnetic Beads, cDNA Library Assay, Plasmid Preparation, Sonication, Ligation, DNA Sequencing, RNA Sequencing Assay

12) Product Images from "Synthetic single domain antibodies for the conformational trapping of membrane proteins"

Article Title: Synthetic single domain antibodies for the conformational trapping of membrane proteins

Journal: eLife

doi: 10.7554/eLife.34317

FX cloning vector series for phage display and purification of sybodies and nanobodies. Sybody pools from ribosome display (or nanobodies from immunized camelids) are amplified with primers containing restriction sites of Type IIS enzyme BspQI (isoschizomer of SapI) to generate AGT and GCA overhangs. BspQI restriction sites generating the same overhangs were introduced into the backbones of vector pDX_init for phage display and pSb_init for periplasmatic expression and attachment of Myc- and His-tag. Note that in pDX_init and pSb_init the BspQI restriction sites are part of the sybody open reading frame. Finally, sybodies/nanobodies are sub-cloned from pSb_init to the destiny vectors pBXNPH3 or pBXNPHM3 for periplasmic expression. Tag-less sybodies/nanobodies for structural biology purposes can be obtained by 3C protease cleavage. Importantly, the vector series permits for PCR-free subcloning once the sybodies have been inserted into phage display vector pDX_init. The vectors were made available through Addgene (for Addgene IDs, see Table 3 ).
Figure Legend Snippet: FX cloning vector series for phage display and purification of sybodies and nanobodies. Sybody pools from ribosome display (or nanobodies from immunized camelids) are amplified with primers containing restriction sites of Type IIS enzyme BspQI (isoschizomer of SapI) to generate AGT and GCA overhangs. BspQI restriction sites generating the same overhangs were introduced into the backbones of vector pDX_init for phage display and pSb_init for periplasmatic expression and attachment of Myc- and His-tag. Note that in pDX_init and pSb_init the BspQI restriction sites are part of the sybody open reading frame. Finally, sybodies/nanobodies are sub-cloned from pSb_init to the destiny vectors pBXNPH3 or pBXNPHM3 for periplasmic expression. Tag-less sybodies/nanobodies for structural biology purposes can be obtained by 3C protease cleavage. Importantly, the vector series permits for PCR-free subcloning once the sybodies have been inserted into phage display vector pDX_init. The vectors were made available through Addgene (for Addgene IDs, see Table 3 ).

Techniques Used: Clone Assay, Plasmid Preparation, Purification, Amplification, Expressing, Polymerase Chain Reaction, Subcloning

13) Product Images from "Cloning and characterization of MAP2191 gene, a mammalian cell entry antigen of Mycobacterium avium subspecies paratuberculosis"

Article Title: Cloning and characterization of MAP2191 gene, a mammalian cell entry antigen of Mycobacterium avium subspecies paratuberculosis

Journal: Molecular Biology Research Communications

doi: 10.22099/mbrc.2018.30979.1354

(A) Restriction digestion of confirmed colony PCR positive pZ57R/T mce -whole clones: lane M: 1kb DNA ladder (#SM0313, Fermentas), lanes 1, 2 and 3: Positive clones of PTZ57R/T mce -whole plasmid digested with Bam HI and Hin dIII enzymes; Lanes 5, 6 and 7: Plasmid isolated of positive PTZ57R/T mce -whole clones. (B) Restriction Digestion of confirmed colony PCR positive pET28a mce -whole clones; lane M: 1kb DNA ladder (#SM0313, Fermentas),lanes 2 and 4: Positive clones of pET28a mce -whole plasmid.
Figure Legend Snippet: (A) Restriction digestion of confirmed colony PCR positive pZ57R/T mce -whole clones: lane M: 1kb DNA ladder (#SM0313, Fermentas), lanes 1, 2 and 3: Positive clones of PTZ57R/T mce -whole plasmid digested with Bam HI and Hin dIII enzymes; Lanes 5, 6 and 7: Plasmid isolated of positive PTZ57R/T mce -whole clones. (B) Restriction Digestion of confirmed colony PCR positive pET28a mce -whole clones; lane M: 1kb DNA ladder (#SM0313, Fermentas),lanes 2 and 4: Positive clones of pET28a mce -whole plasmid.

Techniques Used: Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Isolation

14) Product Images from "Human COX7A2L Regulates Complex III Biogenesis and Promotes Supercomplex Organization Remodeling without Affecting Mitochondrial Bioenergetics"

Article Title: Human COX7A2L Regulates Complex III Biogenesis and Promotes Supercomplex Organization Remodeling without Affecting Mitochondrial Bioenergetics

Journal: Cell reports

doi: 10.1016/j.celrep.2018.10.058

The Short-COX7A2L Variant Binds to CIII2 but Does Not Rescue SC III2+IV Assembly in COX7A2L-KO Cells Control HEK293T cells (WT) and both COX7A2L - KO (KO1 and KO2) clones carrying an empty vector (ev) or constructs to overexpress COX7A2L-Myc- DDK (long) or short-COX7A2L-Myc-DDK (short) were used in the following experiments. (A) SDS-PAGE followed by immunoblotting to estimate steady-state levels of endogenous COX7A2L (~12.6 kDa) from exogenous COX7A2L-Myc-DDK (~16.2 kDa). Membranes were also incubated with antibodies that recognize the indicated OXPHOS subunits. (B). 2D-BN/SDS-PAGE and immunoblotting using digitonin-solubilized mitochondrial extracts (detergent/protein ratio, 4:1) and the indicated antibodies. (C and D) BN-PAGE followed by immunoblotting in digitonized whole-cell extracts (C) or by CI-IGA and CIV-IGA assays and/or immunoblotting in digitonized isolated mitochondria (D). Membranes were incubated with the indicated antibodies. I+III 2 +IV n , SCs containing CI, CIII 2 , and CIV. I+III 2 , SC containing CI and CIII 2 . III 2 +IV, SC containing CIII 2 and CIV. III 2 , complex III dimer (CIII 2 ). IV, complex IV; IV 2 , complex IV dimer (CIV 2 ); II, complex II. Subcomplexes that contain COX1 are indicated as subCOX1. In (D), an unidentified band running a bit faster than the SC III 2 +IV cross-reacting with the COX5B antibody (Ab) (but not with the CORE2 Ab) is indicated with an asterisk.
Figure Legend Snippet: The Short-COX7A2L Variant Binds to CIII2 but Does Not Rescue SC III2+IV Assembly in COX7A2L-KO Cells Control HEK293T cells (WT) and both COX7A2L - KO (KO1 and KO2) clones carrying an empty vector (ev) or constructs to overexpress COX7A2L-Myc- DDK (long) or short-COX7A2L-Myc-DDK (short) were used in the following experiments. (A) SDS-PAGE followed by immunoblotting to estimate steady-state levels of endogenous COX7A2L (~12.6 kDa) from exogenous COX7A2L-Myc-DDK (~16.2 kDa). Membranes were also incubated with antibodies that recognize the indicated OXPHOS subunits. (B). 2D-BN/SDS-PAGE and immunoblotting using digitonin-solubilized mitochondrial extracts (detergent/protein ratio, 4:1) and the indicated antibodies. (C and D) BN-PAGE followed by immunoblotting in digitonized whole-cell extracts (C) or by CI-IGA and CIV-IGA assays and/or immunoblotting in digitonized isolated mitochondria (D). Membranes were incubated with the indicated antibodies. I+III 2 +IV n , SCs containing CI, CIII 2 , and CIV. I+III 2 , SC containing CI and CIII 2 . III 2 +IV, SC containing CIII 2 and CIV. III 2 , complex III dimer (CIII 2 ). IV, complex IV; IV 2 , complex IV dimer (CIV 2 ); II, complex II. Subcomplexes that contain COX1 are indicated as subCOX1. In (D), an unidentified band running a bit faster than the SC III 2 +IV cross-reacting with the COX5B antibody (Ab) (but not with the CORE2 Ab) is indicated with an asterisk.

Techniques Used: Variant Assay, Clone Assay, Plasmid Preparation, Construct, SDS Page, Incubation, Polyacrylamide Gel Electrophoresis, Isolation

15) Product Images from "Genetic polymorphisms in bone morphogenetic protein receptor type IA gene predisposes individuals to ossification of the posterior longitudinal ligament of the cervical spine via the smad signaling pathway"

Article Title: Genetic polymorphisms in bone morphogenetic protein receptor type IA gene predisposes individuals to ossification of the posterior longitudinal ligament of the cervical spine via the smad signaling pathway

Journal: BMC Musculoskeletal Disorders

doi: 10.1186/s12891-018-1966-1

The BMPR-IA gene coding sequence and pGEM-T/BMPR-IA (WT) vector digested with Hind III/Bam HI enzyme were analyzed by 4% agarose gel electrophoresis, respectively. a The annealed ds-oligos of BMPR-IA gene coding sequence was analyzed by 4% agarose gel electrophoresis. Each ds oligo annealing reactant showed a detectable molecular weight band around 1599 bp, as expected for the length of the designed ds-oligos of BMPR-IA gene. Lane M: Marker, Lane B: BMPR-IA gene.0020 b After the pGEM-T/BMPR-IA (WT) vector had been digested with Hind III/Bam HI enzyme, the fragments for BMPR-IA cDNA (1599 bp) and the pGEM-T vector (3000 bp) were observed in all the PCR products as analyzed by 4% agarose gel electrophoresis, respectively. The results showed that the ds-oligos of the BMPR-IA gene cDNA were ligated into the pGEM-T vector. Lane M: Marker, Lane 1: pGEM-T/BMPR-IA (WT) vector, Lane 2: The pGEM-T vector and BMPR-IA cDNA
Figure Legend Snippet: The BMPR-IA gene coding sequence and pGEM-T/BMPR-IA (WT) vector digested with Hind III/Bam HI enzyme were analyzed by 4% agarose gel electrophoresis, respectively. a The annealed ds-oligos of BMPR-IA gene coding sequence was analyzed by 4% agarose gel electrophoresis. Each ds oligo annealing reactant showed a detectable molecular weight band around 1599 bp, as expected for the length of the designed ds-oligos of BMPR-IA gene. Lane M: Marker, Lane B: BMPR-IA gene.0020 b After the pGEM-T/BMPR-IA (WT) vector had been digested with Hind III/Bam HI enzyme, the fragments for BMPR-IA cDNA (1599 bp) and the pGEM-T vector (3000 bp) were observed in all the PCR products as analyzed by 4% agarose gel electrophoresis, respectively. The results showed that the ds-oligos of the BMPR-IA gene cDNA were ligated into the pGEM-T vector. Lane M: Marker, Lane 1: pGEM-T/BMPR-IA (WT) vector, Lane 2: The pGEM-T vector and BMPR-IA cDNA

Techniques Used: IA, Sequencing, Plasmid Preparation, Agarose Gel Electrophoresis, Molecular Weight, Marker, Polymerase Chain Reaction

16) Product Images from "Synthetic single domain antibodies for the conformational trapping of membrane proteins"

Article Title: Synthetic single domain antibodies for the conformational trapping of membrane proteins

Journal: eLife

doi: 10.7554/eLife.34317

FX cloning vector series for phage display and purification of sybodies and nanobodies. Sybody pools from ribosome display (or nanobodies from immunized camelids) are amplified with primers containing restriction sites of Type IIS enzyme BspQI (isoschizomer of SapI) to generate AGT and GCA overhangs. BspQI restriction sites generating the same overhangs were introduced into the backbones of vector pDX_init for phage display and pSb_init for periplasmatic expression and attachment of Myc- and His-tag. Note that in pDX_init and pSb_init the BspQI restriction sites are part of the sybody open reading frame. Finally, sybodies/nanobodies are sub-cloned from pSb_init to the destiny vectors pBXNPH3 or pBXNPHM3 for periplasmic expression. Tag-less sybodies/nanobodies for structural biology purposes can be obtained by 3C protease cleavage. Importantly, the vector series permits for PCR-free subcloning once the sybodies have been inserted into phage display vector pDX_init. The vectors were made available through Addgene (for Addgene IDs, see Table 3 ).
Figure Legend Snippet: FX cloning vector series for phage display and purification of sybodies and nanobodies. Sybody pools from ribosome display (or nanobodies from immunized camelids) are amplified with primers containing restriction sites of Type IIS enzyme BspQI (isoschizomer of SapI) to generate AGT and GCA overhangs. BspQI restriction sites generating the same overhangs were introduced into the backbones of vector pDX_init for phage display and pSb_init for periplasmatic expression and attachment of Myc- and His-tag. Note that in pDX_init and pSb_init the BspQI restriction sites are part of the sybody open reading frame. Finally, sybodies/nanobodies are sub-cloned from pSb_init to the destiny vectors pBXNPH3 or pBXNPHM3 for periplasmic expression. Tag-less sybodies/nanobodies for structural biology purposes can be obtained by 3C protease cleavage. Importantly, the vector series permits for PCR-free subcloning once the sybodies have been inserted into phage display vector pDX_init. The vectors were made available through Addgene (for Addgene IDs, see Table 3 ).

Techniques Used: Clone Assay, Plasmid Preparation, Purification, Amplification, Expressing, Polymerase Chain Reaction, Subcloning

17) Product Images from "Synthetic single domain antibodies for the conformational trapping of membrane proteins"

Article Title: Synthetic single domain antibodies for the conformational trapping of membrane proteins

Journal: eLife

doi: 10.7554/eLife.34317

FX cloning vector series for phage display and purification of sybodies and nanobodies. Sybody pools from ribosome display (or nanobodies from immunized camelids) are amplified with primers containing restriction sites of Type IIS enzyme BspQI (isoschizomer of SapI) to generate AGT and GCA overhangs. BspQI restriction sites generating the same overhangs were introduced into the backbones of vector pDX_init for phage display and pSb_init for periplasmatic expression and attachment of Myc- and His-tag. Note that in pDX_init and pSb_init the BspQI restriction sites are part of the sybody open reading frame. Finally, sybodies/nanobodies are sub-cloned from pSb_init to the destiny vectors pBXNPH3 or pBXNPHM3 for periplasmic expression. Tag-less sybodies/nanobodies for structural biology purposes can be obtained by 3C protease cleavage. Importantly, the vector series permits for PCR-free subcloning once the sybodies have been inserted into phage display vector pDX_init. The vectors were made available through Addgene (for Addgene IDs, see Table 3 ).
Figure Legend Snippet: FX cloning vector series for phage display and purification of sybodies and nanobodies. Sybody pools from ribosome display (or nanobodies from immunized camelids) are amplified with primers containing restriction sites of Type IIS enzyme BspQI (isoschizomer of SapI) to generate AGT and GCA overhangs. BspQI restriction sites generating the same overhangs were introduced into the backbones of vector pDX_init for phage display and pSb_init for periplasmatic expression and attachment of Myc- and His-tag. Note that in pDX_init and pSb_init the BspQI restriction sites are part of the sybody open reading frame. Finally, sybodies/nanobodies are sub-cloned from pSb_init to the destiny vectors pBXNPH3 or pBXNPHM3 for periplasmic expression. Tag-less sybodies/nanobodies for structural biology purposes can be obtained by 3C protease cleavage. Importantly, the vector series permits for PCR-free subcloning once the sybodies have been inserted into phage display vector pDX_init. The vectors were made available through Addgene (for Addgene IDs, see Table 3 ).

Techniques Used: Clone Assay, Plasmid Preparation, Purification, Amplification, Expressing, Polymerase Chain Reaction, Subcloning

18) Product Images from "Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes"

Article Title: Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0107351

Full-length enrichment for library cloning and next generation sequencing (NGS). Full-length (blue line with 5′ cap) or truncated (short blue line without 5′ cap) mRNAs were reverse transcribed into first-strand cDNA using oligo-dT primers (red arrow). The mRNA:cDNA hybrid was treated with RNase I (scissor) to remove the single-stranded RNA that was not fully extended by the first-strand cDNA, followed by selection for full-length transcripts using Cap-antibody magnetic beads to enrich the full-length mRNA:cDNA. The full-length single-stranded DNA (FLssDNA) was eluted from beads and used for both cDNA library cloning (lower left) and NGS (lower right). For full-length library cloning, a double-stranded adaptor (green) was linked to the 5′ end of ssDNA. Second-strand cDNA synthesis was then carried out, followed by cloning into a vector. For NGS, the full-length enriched ssDNA was fragmented by sonication to target fragments in the range of 200–400 bp, followed by ligation of the double-stranded DNA sequencing adaptor mixture (purple) to 3′ and 5′ ends of ssDNA. To maintain the complexity of the library while enriching the full-length cDNA for NGS, the original polyA mRNA was also fragmented using RNAse III, followed by ligation of the double-stranded RNA sequencing adaptor mixture (brown) to 3′ and 5′ ends of mRNA. After first- and second-strand synthesis, the polyA and capped mRNA and polyA and non-capped mRNA samples were mixed in a 3∶1 ratio and applied to the downstream NGS procedure.
Figure Legend Snippet: Full-length enrichment for library cloning and next generation sequencing (NGS). Full-length (blue line with 5′ cap) or truncated (short blue line without 5′ cap) mRNAs were reverse transcribed into first-strand cDNA using oligo-dT primers (red arrow). The mRNA:cDNA hybrid was treated with RNase I (scissor) to remove the single-stranded RNA that was not fully extended by the first-strand cDNA, followed by selection for full-length transcripts using Cap-antibody magnetic beads to enrich the full-length mRNA:cDNA. The full-length single-stranded DNA (FLssDNA) was eluted from beads and used for both cDNA library cloning (lower left) and NGS (lower right). For full-length library cloning, a double-stranded adaptor (green) was linked to the 5′ end of ssDNA. Second-strand cDNA synthesis was then carried out, followed by cloning into a vector. For NGS, the full-length enriched ssDNA was fragmented by sonication to target fragments in the range of 200–400 bp, followed by ligation of the double-stranded DNA sequencing adaptor mixture (purple) to 3′ and 5′ ends of ssDNA. To maintain the complexity of the library while enriching the full-length cDNA for NGS, the original polyA mRNA was also fragmented using RNAse III, followed by ligation of the double-stranded RNA sequencing adaptor mixture (brown) to 3′ and 5′ ends of mRNA. After first- and second-strand synthesis, the polyA and capped mRNA and polyA and non-capped mRNA samples were mixed in a 3∶1 ratio and applied to the downstream NGS procedure.

Techniques Used: Clone Assay, Next-Generation Sequencing, Selection, Magnetic Beads, cDNA Library Assay, Plasmid Preparation, Sonication, Ligation, DNA Sequencing, RNA Sequencing Assay

19) Product Images from "Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription"

Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

Journal: Nucleic Acids Research

doi: 10.1093/nar/gni064

Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.
Figure Legend Snippet: Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.

Techniques Used: Synthesized, In Vitro, Plasmid Preparation, Agarose Gel Electrophoresis

20) Product Images from "Cloning and characterization of MAP2191 gene, a mammalian cell entry antigen of Mycobacterium avium subspecies paratuberculosis"

Article Title: Cloning and characterization of MAP2191 gene, a mammalian cell entry antigen of Mycobacterium avium subspecies paratuberculosis

Journal: Molecular Biology Research Communications

doi: 10.22099/mbrc.2018.30979.1354

(A) Restriction digestion of confirmed colony PCR positive pZ57R/T mce -whole clones: lane M: 1kb DNA ladder (#SM0313, Fermentas), lanes 1, 2 and 3: Positive clones of PTZ57R/T mce -whole plasmid digested with Bam HI and Hin dIII enzymes; Lanes 5, 6 and 7: Plasmid isolated of positive PTZ57R/T mce -whole clones. (B) Restriction Digestion of confirmed colony PCR positive pET28a mce -whole clones; lane M: 1kb DNA ladder (#SM0313, Fermentas),lanes 2 and 4: Positive clones of pET28a mce -whole plasmid.
Figure Legend Snippet: (A) Restriction digestion of confirmed colony PCR positive pZ57R/T mce -whole clones: lane M: 1kb DNA ladder (#SM0313, Fermentas), lanes 1, 2 and 3: Positive clones of PTZ57R/T mce -whole plasmid digested with Bam HI and Hin dIII enzymes; Lanes 5, 6 and 7: Plasmid isolated of positive PTZ57R/T mce -whole clones. (B) Restriction Digestion of confirmed colony PCR positive pET28a mce -whole clones; lane M: 1kb DNA ladder (#SM0313, Fermentas),lanes 2 and 4: Positive clones of pET28a mce -whole plasmid.

Techniques Used: Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Isolation

(A) Analysis of Mce-whole Protein expressed by SDS PAGE. (Left to Right), lane M: Protein molecular weight marker (SL7012, CinnaGen), lane 1: pET28a.Mce.whole Elution 1, lane 2: pET28a.Mce.whole Supernatant, lane 3: pET28a.Mce.whole Pellet, lane 4. pET28a.Mce.whole Crude; (B) Western blot analysis (from left to right) lane M: Protein molecular weight marker (SL7012, CinnaGen), lane 1: pET28a.Mce.whole Elution 1, lane 2: p1ET28a.Mce.whole Supernatant, lane 3: pET28a.Mce.whole Pellet, lane 4. pET28a.Mce.whole Crude.
Figure Legend Snippet: (A) Analysis of Mce-whole Protein expressed by SDS PAGE. (Left to Right), lane M: Protein molecular weight marker (SL7012, CinnaGen), lane 1: pET28a.Mce.whole Elution 1, lane 2: pET28a.Mce.whole Supernatant, lane 3: pET28a.Mce.whole Pellet, lane 4. pET28a.Mce.whole Crude; (B) Western blot analysis (from left to right) lane M: Protein molecular weight marker (SL7012, CinnaGen), lane 1: pET28a.Mce.whole Elution 1, lane 2: p1ET28a.Mce.whole Supernatant, lane 3: pET28a.Mce.whole Pellet, lane 4. pET28a.Mce.whole Crude.

Techniques Used: SDS Page, Molecular Weight, Marker, Western Blot

21) Product Images from "Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription"

Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

Journal: Nucleic Acids Research

doi: 10.1093/nar/gni064

Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.
Figure Legend Snippet: Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.

Techniques Used: Synthesized, In Vitro, Plasmid Preparation, Agarose Gel Electrophoresis

22) Product Images from "Genetic polymorphisms in bone morphogenetic protein receptor type IA gene predisposes individuals to ossification of the posterior longitudinal ligament of the cervical spine via the smad signaling pathway"

Article Title: Genetic polymorphisms in bone morphogenetic protein receptor type IA gene predisposes individuals to ossification of the posterior longitudinal ligament of the cervical spine via the smad signaling pathway

Journal: BMC Musculoskeletal Disorders

doi: 10.1186/s12891-018-1966-1

The BMPR-IA gene coding sequence and pGEM-T/BMPR-IA (WT) vector digested with Hind III/Bam HI enzyme were analyzed by 4% agarose gel electrophoresis, respectively. a The annealed ds-oligos of BMPR-IA gene coding sequence was analyzed by 4% agarose gel electrophoresis. Each ds oligo annealing reactant showed a detectable molecular weight band around 1599 bp, as expected for the length of the designed ds-oligos of BMPR-IA gene. Lane M: Marker, Lane B: BMPR-IA gene.0020 b After the pGEM-T/BMPR-IA (WT) vector had been digested with Hind III/Bam HI enzyme, the fragments for BMPR-IA cDNA (1599 bp) and the pGEM-T vector (3000 bp) were observed in all the PCR products as analyzed by 4% agarose gel electrophoresis, respectively. The results showed that the ds-oligos of the BMPR-IA gene cDNA were ligated into the pGEM-T vector. Lane M: Marker, Lane 1: pGEM-T/BMPR-IA (WT) vector, Lane 2: The pGEM-T vector and BMPR-IA cDNA
Figure Legend Snippet: The BMPR-IA gene coding sequence and pGEM-T/BMPR-IA (WT) vector digested with Hind III/Bam HI enzyme were analyzed by 4% agarose gel electrophoresis, respectively. a The annealed ds-oligos of BMPR-IA gene coding sequence was analyzed by 4% agarose gel electrophoresis. Each ds oligo annealing reactant showed a detectable molecular weight band around 1599 bp, as expected for the length of the designed ds-oligos of BMPR-IA gene. Lane M: Marker, Lane B: BMPR-IA gene.0020 b After the pGEM-T/BMPR-IA (WT) vector had been digested with Hind III/Bam HI enzyme, the fragments for BMPR-IA cDNA (1599 bp) and the pGEM-T vector (3000 bp) were observed in all the PCR products as analyzed by 4% agarose gel electrophoresis, respectively. The results showed that the ds-oligos of the BMPR-IA gene cDNA were ligated into the pGEM-T vector. Lane M: Marker, Lane 1: pGEM-T/BMPR-IA (WT) vector, Lane 2: The pGEM-T vector and BMPR-IA cDNA

Techniques Used: IA, Sequencing, Plasmid Preparation, Agarose Gel Electrophoresis, Molecular Weight, Marker, Polymerase Chain Reaction

23) Product Images from "Removal of PCR Error Products and Unincorporated Primers by Metal-Chelate Affinity Chromatography"

Article Title: Removal of PCR Error Products and Unincorporated Primers by Metal-Chelate Affinity Chromatography

Journal: PLoS ONE

doi: 10.1371/journal.pone.0014512

Cu 2+ -IMAC purification of PCR product mixture from amplifying a region of lambda bacteriophage genomic DNA. ( A ) Gel picture showing Cu 2+ -IMAC purification of PCR product mixture from amplification of a 280 bp region of lambda bacteriophage genomic DNA. Lane 1: 5 µL of unpurified PCR product; Lane 2: 5 µL of purified PCR product, flow-through after direct application of PCR product mixture to Cu 2+ -IMAC column; Lanes 3–5: 5 µL of consecutive 20 µL column washes with 250 mM NaCl, 20 mM HEPES, pH 7.0; Lane 6: 5 µL of first elution with 20 µL 500 mM imidazole in 250 mM NaCl, 20 mM HEPES, pH 7.0; Lanes 7–9: 5 µL of first, second and third elutions, respectively with 20 µL 500 mM imidazole in 250 mM NaCl, 20 mM HEPES, pH 7.0. Lanes 7–9 were concentrated 16-fold by ethanol precipitation to enhance sensitivity. ( B ) Expanded views of 280 bp product from selected lanes of Figure 2A . Lane 1: Unpurified PCR product, corresponding to Lane 1 of Figure 2A ; Lane 2: Purified flow-through PCR product, corresponding to Lane 2 of Figure 2A ; Lane 3: First wash with 250 mM NaCl, 20 mM HEPES, pH 7.0, corresponding to Lane 3 of Figure 2A ; Lane 6: First elution with 500 mM imidazole in 250 mM NaCl, 20 mM HEPES, pH 7.0, corresponding to Lane 6 of Figure 2A ; Lane 7: 16 times concentrated product of first imidazole elution, corresponding to Lane 7 of Figure 2A .
Figure Legend Snippet: Cu 2+ -IMAC purification of PCR product mixture from amplifying a region of lambda bacteriophage genomic DNA. ( A ) Gel picture showing Cu 2+ -IMAC purification of PCR product mixture from amplification of a 280 bp region of lambda bacteriophage genomic DNA. Lane 1: 5 µL of unpurified PCR product; Lane 2: 5 µL of purified PCR product, flow-through after direct application of PCR product mixture to Cu 2+ -IMAC column; Lanes 3–5: 5 µL of consecutive 20 µL column washes with 250 mM NaCl, 20 mM HEPES, pH 7.0; Lane 6: 5 µL of first elution with 20 µL 500 mM imidazole in 250 mM NaCl, 20 mM HEPES, pH 7.0; Lanes 7–9: 5 µL of first, second and third elutions, respectively with 20 µL 500 mM imidazole in 250 mM NaCl, 20 mM HEPES, pH 7.0. Lanes 7–9 were concentrated 16-fold by ethanol precipitation to enhance sensitivity. ( B ) Expanded views of 280 bp product from selected lanes of Figure 2A . Lane 1: Unpurified PCR product, corresponding to Lane 1 of Figure 2A ; Lane 2: Purified flow-through PCR product, corresponding to Lane 2 of Figure 2A ; Lane 3: First wash with 250 mM NaCl, 20 mM HEPES, pH 7.0, corresponding to Lane 3 of Figure 2A ; Lane 6: First elution with 500 mM imidazole in 250 mM NaCl, 20 mM HEPES, pH 7.0, corresponding to Lane 6 of Figure 2A ; Lane 7: 16 times concentrated product of first imidazole elution, corresponding to Lane 7 of Figure 2A .

Techniques Used: Purification, Polymerase Chain Reaction, Amplification, Flow Cytometry, Ethanol Precipitation

24) Product Images from "Detection of Circulating hcmv-miR-UL112-3p in Patients with Glioblastoma, Rheumatoid Arthritis, Diabetes Mellitus and Healthy Controls"

Article Title: Detection of Circulating hcmv-miR-UL112-3p in Patients with Glioblastoma, Rheumatoid Arthritis, Diabetes Mellitus and Healthy Controls

Journal: PLoS ONE

doi: 10.1371/journal.pone.0113740

Confirmation of a cloned hcmv-miRUL112-3p TaqMan PCR amplicon from in vitro infected HCMV lung fibroblasts (Positive Control): Alignment of sequences (from above) shows the PCR2.1 TOPO empty plasmid, hcmv-miR-UL112 sequence from miRBase database and last two sequences confirms the PCR product was inserted into the PCR2.1 TOPO plasmid. The lower part represents the chromatogram for the recombinant plasmid.
Figure Legend Snippet: Confirmation of a cloned hcmv-miRUL112-3p TaqMan PCR amplicon from in vitro infected HCMV lung fibroblasts (Positive Control): Alignment of sequences (from above) shows the PCR2.1 TOPO empty plasmid, hcmv-miR-UL112 sequence from miRBase database and last two sequences confirms the PCR product was inserted into the PCR2.1 TOPO plasmid. The lower part represents the chromatogram for the recombinant plasmid.

Techniques Used: Clone Assay, Polymerase Chain Reaction, Amplification, In Vitro, Infection, Positive Control, Plasmid Preparation, Sequencing, Recombinant

25) Product Images from "Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription"

Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

Journal: Nucleic Acids Research

doi: 10.1093/nar/gni064

Agarose gel electrophoresis of RDA products ( A ) and its hybridized autoradiogram ( B ). In vitro transcribed RNA and total cellular RNA were mixed as described in the legend to Figure 2 . Double-stranded cDNAs were synthesized and subjected to RDA as described in Materials and Methods. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide ( A ), blotted onto a nylon membrane and hybridized with 32 P-labelled pCIneo ( B ). Positions and sizes (bp) of markers are present on the left.
Figure Legend Snippet: Agarose gel electrophoresis of RDA products ( A ) and its hybridized autoradiogram ( B ). In vitro transcribed RNA and total cellular RNA were mixed as described in the legend to Figure 2 . Double-stranded cDNAs were synthesized and subjected to RDA as described in Materials and Methods. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide ( A ), blotted onto a nylon membrane and hybridized with 32 P-labelled pCIneo ( B ). Positions and sizes (bp) of markers are present on the left.

Techniques Used: Agarose Gel Electrophoresis, In Vitro, Synthesized, Amplification, Staining

Agarose gel electrophoresis of RDA products from RNA extracted from bovine parainfluenza virus 3-infected cells. Double-stranded cDNA was synthesized from RNA of bovine parainfluenza virus 3-infected MDBK cells and subjected to RDA. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide. RDA product from the uninfected control cells was used as a negative control.
Figure Legend Snippet: Agarose gel electrophoresis of RDA products from RNA extracted from bovine parainfluenza virus 3-infected cells. Double-stranded cDNA was synthesized from RNA of bovine parainfluenza virus 3-infected MDBK cells and subjected to RDA. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide. RDA product from the uninfected control cells was used as a negative control.

Techniques Used: Agarose Gel Electrophoresis, Infection, Synthesized, Amplification, Staining, Negative Control

Agarose gel electrophoresis of RDA products with PCR products used for probes for hybridization ( A ) and a hybridized fluorogram ( B ). RNA was extracted from SARS-CoV-infected cells and subjected to RDA according to the method described in Materials and Methods. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gels and blotted on a Nylon membrane. The membrane was then cut into slits that contained the lane showing the presence of DNA. On the other hand, the PCR fragments predicted to be amplified in the RDA reaction were amplified and subsequently ascertained by agarose gel electrophoresis (A). The amplified genomic fragments of SARS-CoV were Dig-labelled and used as probes for hybridization to each slit of the Nylon membrane containing the RDA product. Hybridization was performed in separate hybridization bags. After washing with 1× SSC and 0.1% SDS solution, the hybridized probes were detected on a fluorogram (B). Positions and sizes (bp) of markers are present on the left.
Figure Legend Snippet: Agarose gel electrophoresis of RDA products with PCR products used for probes for hybridization ( A ) and a hybridized fluorogram ( B ). RNA was extracted from SARS-CoV-infected cells and subjected to RDA according to the method described in Materials and Methods. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gels and blotted on a Nylon membrane. The membrane was then cut into slits that contained the lane showing the presence of DNA. On the other hand, the PCR fragments predicted to be amplified in the RDA reaction were amplified and subsequently ascertained by agarose gel electrophoresis (A). The amplified genomic fragments of SARS-CoV were Dig-labelled and used as probes for hybridization to each slit of the Nylon membrane containing the RDA product. Hybridization was performed in separate hybridization bags. After washing with 1× SSC and 0.1% SDS solution, the hybridized probes were detected on a fluorogram (B). Positions and sizes (bp) of markers are present on the left.

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Hybridization, Infection, Amplification

Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.
Figure Legend Snippet: Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.

Techniques Used: Synthesized, In Vitro, Plasmid Preparation, Agarose Gel Electrophoresis

26) Product Images from "Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes"

Article Title: Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0107351

Full-length enrichment for library cloning and next generation sequencing (NGS). Full-length (blue line with 5′ cap) or truncated (short blue line without 5′ cap) mRNAs were reverse transcribed into first-strand cDNA using oligo-dT primers (red arrow). The mRNA:cDNA hybrid was treated with RNase I (scissor) to remove the single-stranded RNA that was not fully extended by the first-strand cDNA, followed by selection for full-length transcripts using Cap-antibody magnetic beads to enrich the full-length mRNA:cDNA. The full-length single-stranded DNA (FLssDNA) was eluted from beads and used for both cDNA library cloning (lower left) and NGS (lower right). For full-length library cloning, a double-stranded adaptor (green) was linked to the 5′ end of ssDNA. Second-strand cDNA synthesis was then carried out, followed by cloning into a vector. For NGS, the full-length enriched ssDNA was fragmented by sonication to target fragments in the range of 200–400 bp, followed by ligation of the double-stranded DNA sequencing adaptor mixture (purple) to 3′ and 5′ ends of ssDNA. To maintain the complexity of the library while enriching the full-length cDNA for NGS, the original polyA mRNA was also fragmented using RNAse III, followed by ligation of the double-stranded RNA sequencing adaptor mixture (brown) to 3′ and 5′ ends of mRNA. After first- and second-strand synthesis, the polyA and capped mRNA and polyA and non-capped mRNA samples were mixed in a 3∶1 ratio and applied to the downstream NGS procedure.
Figure Legend Snippet: Full-length enrichment for library cloning and next generation sequencing (NGS). Full-length (blue line with 5′ cap) or truncated (short blue line without 5′ cap) mRNAs were reverse transcribed into first-strand cDNA using oligo-dT primers (red arrow). The mRNA:cDNA hybrid was treated with RNase I (scissor) to remove the single-stranded RNA that was not fully extended by the first-strand cDNA, followed by selection for full-length transcripts using Cap-antibody magnetic beads to enrich the full-length mRNA:cDNA. The full-length single-stranded DNA (FLssDNA) was eluted from beads and used for both cDNA library cloning (lower left) and NGS (lower right). For full-length library cloning, a double-stranded adaptor (green) was linked to the 5′ end of ssDNA. Second-strand cDNA synthesis was then carried out, followed by cloning into a vector. For NGS, the full-length enriched ssDNA was fragmented by sonication to target fragments in the range of 200–400 bp, followed by ligation of the double-stranded DNA sequencing adaptor mixture (purple) to 3′ and 5′ ends of ssDNA. To maintain the complexity of the library while enriching the full-length cDNA for NGS, the original polyA mRNA was also fragmented using RNAse III, followed by ligation of the double-stranded RNA sequencing adaptor mixture (brown) to 3′ and 5′ ends of mRNA. After first- and second-strand synthesis, the polyA and capped mRNA and polyA and non-capped mRNA samples were mixed in a 3∶1 ratio and applied to the downstream NGS procedure.

Techniques Used: Clone Assay, Next-Generation Sequencing, Selection, Magnetic Beads, cDNA Library Assay, Plasmid Preparation, Sonication, Ligation, DNA Sequencing, RNA Sequencing Assay

27) Product Images from "Modulation of the transcription factor NF-κB in antigen-presenting cells by bovine respiratory syncytial virus small hydrophobic protein"

Article Title: Modulation of the transcription factor NF-κB in antigen-presenting cells by bovine respiratory syncytial virus small hydrophobic protein

Journal: The Journal of General Virology

doi: 10.1099/jgv.0.000855

Expression of recombinant SH-V5/His 6 in bovine CD14 + . Primary bovine CD14 + cells were electroporated with pCDNA6 SH-V5/His6 (a) or pCDNA-GFP (b) or mock-electroporated (a and b). After 24 h in culture, cells were permeabilized and intracellular expression of SH-V5/His 6 was detected using phycoerythrin (PE)-conjugated anti-V5; PE and GFP expression were measured by flow cytometry. Black histograms show autofluorescence of mock-electroporated cells stained with PE-conjugated anti-V5 (a) or autofluorescence alone (b); the grey-filled histogram shows fluorescence of electroporated cells stained with PE-conjugated anti-V5 (a) or GFP fluorescence (b). Figure representative of three experiments analysed in duplicate.
Figure Legend Snippet: Expression of recombinant SH-V5/His 6 in bovine CD14 + . Primary bovine CD14 + cells were electroporated with pCDNA6 SH-V5/His6 (a) or pCDNA-GFP (b) or mock-electroporated (a and b). After 24 h in culture, cells were permeabilized and intracellular expression of SH-V5/His 6 was detected using phycoerythrin (PE)-conjugated anti-V5; PE and GFP expression were measured by flow cytometry. Black histograms show autofluorescence of mock-electroporated cells stained with PE-conjugated anti-V5 (a) or autofluorescence alone (b); the grey-filled histogram shows fluorescence of electroporated cells stained with PE-conjugated anti-V5 (a) or GFP fluorescence (b). Figure representative of three experiments analysed in duplicate.

Techniques Used: Expressing, Recombinant, Flow Cytometry, Cytometry, Staining, Fluorescence

28) Product Images from "Disruption of tetR type regulator adeN by mobile genetic element confers elevated virulence in Acinetobacter baumannii"

Article Title: Disruption of tetR type regulator adeN by mobile genetic element confers elevated virulence in Acinetobacter baumannii

Journal: Virulence

doi: 10.1080/21505594.2017.1322240

Survival Plot of Galleria mellonella larvae infected with the adeN knockout, wild type and complemented strains. PKAB07 and Δ adeN strains were found to be hyper- virulent and killed relatively higher number of larvae when compared with controls, ATCC 17978 and PBS. The virulence was compromised when G. mellonella infected with the complemented strain of Δ adeN with pWH1266:: adeN plasmid.
Figure Legend Snippet: Survival Plot of Galleria mellonella larvae infected with the adeN knockout, wild type and complemented strains. PKAB07 and Δ adeN strains were found to be hyper- virulent and killed relatively higher number of larvae when compared with controls, ATCC 17978 and PBS. The virulence was compromised when G. mellonella infected with the complemented strain of Δ adeN with pWH1266:: adeN plasmid.

Techniques Used: Infection, Knock-Out, Plasmid Preparation

(A) Co-culturing followed by AO/EB stainingA549 cells co-cultured with A. baumannii strains were added with AO/EB viability check stain and observed under fluorescent microscope B-2A filter (Nikon Eclipse TS100, Japan). PKAB07 and Δ adeN strains were found to be hyper invasive and induced more cell death as evidenced by the cellular uptake of ethidium bromide by A549 cells (B C) when compared with the uninfected control (A). The virulence was compromised when the ΔadeN strain was complemented with pWH1266:: adeN plasmid (D). Scale bar represents 50µm at 40X magnification.
Figure Legend Snippet: (A) Co-culturing followed by AO/EB stainingA549 cells co-cultured with A. baumannii strains were added with AO/EB viability check stain and observed under fluorescent microscope B-2A filter (Nikon Eclipse TS100, Japan). PKAB07 and Δ adeN strains were found to be hyper invasive and induced more cell death as evidenced by the cellular uptake of ethidium bromide by A549 cells (B C) when compared with the uninfected control (A). The virulence was compromised when the ΔadeN strain was complemented with pWH1266:: adeN plasmid (D). Scale bar represents 50µm at 40X magnification.

Techniques Used: Cell Culture, Staining, Microscopy, Plasmid Preparation

29) Product Images from "Synthetic single domain antibodies for the conformational trapping of membrane proteins"

Article Title: Synthetic single domain antibodies for the conformational trapping of membrane proteins

Journal: eLife

doi: 10.7554/eLife.34317

FX cloning vector series for phage display and purification of sybodies and nanobodies. Sybody pools from ribosome display (or nanobodies from immunized camelids) are amplified with primers containing restriction sites of Type IIS enzyme BspQI (isoschizomer of SapI) to generate AGT and GCA overhangs. BspQI restriction sites generating the same overhangs were introduced into the backbones of vector pDX_init for phage display and pSb_init for periplasmatic expression and attachment of Myc- and His-tag. Note that in pDX_init and pSb_init the BspQI restriction sites are part of the sybody open reading frame. Finally, sybodies/nanobodies are sub-cloned from pSb_init to the destiny vectors pBXNPH3 or pBXNPHM3 for periplasmic expression. Tag-less sybodies/nanobodies for structural biology purposes can be obtained by 3C protease cleavage. Importantly, the vector series permits for PCR-free subcloning once the sybodies have been inserted into phage display vector pDX_init. The vectors were made available through Addgene (for Addgene IDs, see Table 3 ).
Figure Legend Snippet: FX cloning vector series for phage display and purification of sybodies and nanobodies. Sybody pools from ribosome display (or nanobodies from immunized camelids) are amplified with primers containing restriction sites of Type IIS enzyme BspQI (isoschizomer of SapI) to generate AGT and GCA overhangs. BspQI restriction sites generating the same overhangs were introduced into the backbones of vector pDX_init for phage display and pSb_init for periplasmatic expression and attachment of Myc- and His-tag. Note that in pDX_init and pSb_init the BspQI restriction sites are part of the sybody open reading frame. Finally, sybodies/nanobodies are sub-cloned from pSb_init to the destiny vectors pBXNPH3 or pBXNPHM3 for periplasmic expression. Tag-less sybodies/nanobodies for structural biology purposes can be obtained by 3C protease cleavage. Importantly, the vector series permits for PCR-free subcloning once the sybodies have been inserted into phage display vector pDX_init. The vectors were made available through Addgene (for Addgene IDs, see Table 3 ).

Techniques Used: Clone Assay, Plasmid Preparation, Purification, Amplification, Expressing, Polymerase Chain Reaction, Subcloning

30) Product Images from "Genetic polymorphisms in bone morphogenetic protein receptor type IA gene predisposes individuals to ossification of the posterior longitudinal ligament of the cervical spine via the smad signaling pathway"

Article Title: Genetic polymorphisms in bone morphogenetic protein receptor type IA gene predisposes individuals to ossification of the posterior longitudinal ligament of the cervical spine via the smad signaling pathway

Journal: BMC Musculoskeletal Disorders

doi: 10.1186/s12891-018-1966-1

The BMPR-IA gene coding sequence and pGEM-T/BMPR-IA (WT) vector digested with Hind III/Bam HI enzyme were analyzed by 4% agarose gel electrophoresis, respectively. a The annealed ds-oligos of BMPR-IA gene coding sequence was analyzed by 4% agarose gel electrophoresis. Each ds oligo annealing reactant showed a detectable molecular weight band around 1599 bp, as expected for the length of the designed ds-oligos of BMPR-IA gene. Lane M: Marker, Lane B: BMPR-IA gene.0020 b After the pGEM-T/BMPR-IA (WT) vector had been digested with Hind III/Bam HI enzyme, the fragments for BMPR-IA cDNA (1599 bp) and the pGEM-T vector (3000 bp) were observed in all the PCR products as analyzed by 4% agarose gel electrophoresis, respectively. The results showed that the ds-oligos of the BMPR-IA gene cDNA were ligated into the pGEM-T vector. Lane M: Marker, Lane 1: pGEM-T/BMPR-IA (WT) vector, Lane 2: The pGEM-T vector and BMPR-IA cDNA
Figure Legend Snippet: The BMPR-IA gene coding sequence and pGEM-T/BMPR-IA (WT) vector digested with Hind III/Bam HI enzyme were analyzed by 4% agarose gel electrophoresis, respectively. a The annealed ds-oligos of BMPR-IA gene coding sequence was analyzed by 4% agarose gel electrophoresis. Each ds oligo annealing reactant showed a detectable molecular weight band around 1599 bp, as expected for the length of the designed ds-oligos of BMPR-IA gene. Lane M: Marker, Lane B: BMPR-IA gene.0020 b After the pGEM-T/BMPR-IA (WT) vector had been digested with Hind III/Bam HI enzyme, the fragments for BMPR-IA cDNA (1599 bp) and the pGEM-T vector (3000 bp) were observed in all the PCR products as analyzed by 4% agarose gel electrophoresis, respectively. The results showed that the ds-oligos of the BMPR-IA gene cDNA were ligated into the pGEM-T vector. Lane M: Marker, Lane 1: pGEM-T/BMPR-IA (WT) vector, Lane 2: The pGEM-T vector and BMPR-IA cDNA

Techniques Used: IA, Sequencing, Plasmid Preparation, Agarose Gel Electrophoresis, Molecular Weight, Marker, Polymerase Chain Reaction

31) Product Images from "High-throughput profiling of point mutations across the HIV-1 genome"

Article Title: High-throughput profiling of point mutations across the HIV-1 genome

Journal: Retrovirology

doi: 10.1186/s12977-014-0124-6

HIV-1 mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step PCR amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
Figure Legend Snippet: HIV-1 mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step PCR amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.

Techniques Used: Mutagenesis, Next-Generation Sequencing, Sample Prep, Polymerase Chain Reaction, Amplification, Concentration Assay

32) Product Images from "The preparation of HL-60 cells vaccine expressing BCG heat shock protein 70 and detection of its immunogenicity in vitro"

Article Title: The preparation of HL-60 cells vaccine expressing BCG heat shock protein 70 and detection of its immunogenicity in vitro

Journal: Human Vaccines & Immunotherapeutics

doi: 10.4161/hv.21321

Figure 5. Regulation of T cells function (IFN-γ). Purified T cells were co-cultured with HL60-wt, HL60-pDisplay and HL60-HSP70 cells at the radio of 10:1. Cytokine production of IFN-γ from T cells was measured by ELISA. The highest
Figure Legend Snippet: Figure 5. Regulation of T cells function (IFN-γ). Purified T cells were co-cultured with HL60-wt, HL60-pDisplay and HL60-HSP70 cells at the radio of 10:1. Cytokine production of IFN-γ from T cells was measured by ELISA. The highest

Techniques Used: Purification, Cell Culture, Enzyme-linked Immunosorbent Assay

Figure 2. Identification of recombinant plasmid pDisplay-HSP70. Lane 1: DNA marker (DL2000); Lane 2: PCR product of HSP70; Lane 3: plasmid pDisplay digested with BglII and SmaI; Lane 4: HSP70 digested with BglII and SmaI; Lane 5: recombinant plasmid
Figure Legend Snippet: Figure 2. Identification of recombinant plasmid pDisplay-HSP70. Lane 1: DNA marker (DL2000); Lane 2: PCR product of HSP70; Lane 3: plasmid pDisplay digested with BglII and SmaI; Lane 4: HSP70 digested with BglII and SmaI; Lane 5: recombinant plasmid

Techniques Used: Recombinant, Plasmid Preparation, Marker, Polymerase Chain Reaction

33) Product Images from "Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *"

Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.384321

SDS-PAGE analysis of purified DNA polymerase I, DNA ligase I, and the Vsr endonuclease. Purified proteins (3 μg) were resolved on an 11% polyacrylamide gel run in the presence of SDS and stained with Coomassie Blue. Lane 1 , molecular mass standards
Figure Legend Snippet: SDS-PAGE analysis of purified DNA polymerase I, DNA ligase I, and the Vsr endonuclease. Purified proteins (3 μg) were resolved on an 11% polyacrylamide gel run in the presence of SDS and stained with Coomassie Blue. Lane 1 , molecular mass standards

Techniques Used: SDS Page, Purification, Staining

DNA polymerase I and the Vsr endonuclease are sufficient to repair a T:G mismatch. The VSP repair reaction contained 50 ng of covalently closed heteroduplex substrate, 52 n m Vsr endonuclease, and a titration of DNA polymerase I from 467 to 0.2 n m as described
Figure Legend Snippet: DNA polymerase I and the Vsr endonuclease are sufficient to repair a T:G mismatch. The VSP repair reaction contained 50 ng of covalently closed heteroduplex substrate, 52 n m Vsr endonuclease, and a titration of DNA polymerase I from 467 to 0.2 n m as described

Techniques Used: Titration

DNA ligase I seals the nick created by DNA polymerase I nick translation. Repair and ligation reactions were conducted as described under “Materials and Methods” using 200 ng of pUC19-VSR heteroduplex DNA (∼1.2 n m molecules), 10
Figure Legend Snippet: DNA ligase I seals the nick created by DNA polymerase I nick translation. Repair and ligation reactions were conducted as described under “Materials and Methods” using 200 ng of pUC19-VSR heteroduplex DNA (∼1.2 n m molecules), 10

Techniques Used: Nick Translation, Ligation

Vsr-catalyzed nicking of a covalently closed circular DNA substrate. Vsr-dependent nicking reactions were as described under “Materials and Methods.” A , the titration of the Vsr endonuclease was from 153 to 0 n m with each DNA substrate,
Figure Legend Snippet: Vsr-catalyzed nicking of a covalently closed circular DNA substrate. Vsr-dependent nicking reactions were as described under “Materials and Methods.” A , the titration of the Vsr endonuclease was from 153 to 0 n m with each DNA substrate,

Techniques Used: Titration

The impact of DNA ligase I concentration on repair track length. Ligation and repair reactions were as described under “Materials and Methods” using 200 ng of covalently closed pUC19-VSR heteroduplex DNA (∼1.2 n m molecules), 10
Figure Legend Snippet: The impact of DNA ligase I concentration on repair track length. Ligation and repair reactions were as described under “Materials and Methods” using 200 ng of covalently closed pUC19-VSR heteroduplex DNA (∼1.2 n m molecules), 10

Techniques Used: Concentration Assay, Ligation

The Vsr endonuclease incises DNA immediately 5′ to the mismatched thymidine. Vsr nicking reactions were as described under “Materials and Methods.” The Vsr endonuclease was incubated with covalently closed pUC19-VSR heteroduplex
Figure Legend Snippet: The Vsr endonuclease incises DNA immediately 5′ to the mismatched thymidine. Vsr nicking reactions were as described under “Materials and Methods.” The Vsr endonuclease was incubated with covalently closed pUC19-VSR heteroduplex

Techniques Used: Incubation

34) Product Images from "Cloning and characterization of MAP2191 gene, a mammalian cell entry antigen of Mycobacterium avium subspecies paratuberculosis"

Article Title: Cloning and characterization of MAP2191 gene, a mammalian cell entry antigen of Mycobacterium avium subspecies paratuberculosis

Journal: Molecular Biology Research Communications

doi: 10.22099/mbrc.2018.30979.1354

(A) Restriction digestion of confirmed colony PCR positive pZ57R/T mce -whole clones: lane M: 1kb DNA ladder (#SM0313, Fermentas), lanes 1, 2 and 3: Positive clones of PTZ57R/T mce -whole plasmid digested with Bam HI and Hin dIII enzymes; Lanes 5, 6 and 7: Plasmid isolated of positive PTZ57R/T mce -whole clones. (B) Restriction Digestion of confirmed colony PCR positive pET28a mce -whole clones; lane M: 1kb DNA ladder (#SM0313, Fermentas),lanes 2 and 4: Positive clones of pET28a mce -whole plasmid.
Figure Legend Snippet: (A) Restriction digestion of confirmed colony PCR positive pZ57R/T mce -whole clones: lane M: 1kb DNA ladder (#SM0313, Fermentas), lanes 1, 2 and 3: Positive clones of PTZ57R/T mce -whole plasmid digested with Bam HI and Hin dIII enzymes; Lanes 5, 6 and 7: Plasmid isolated of positive PTZ57R/T mce -whole clones. (B) Restriction Digestion of confirmed colony PCR positive pET28a mce -whole clones; lane M: 1kb DNA ladder (#SM0313, Fermentas),lanes 2 and 4: Positive clones of pET28a mce -whole plasmid.

Techniques Used: Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Isolation

35) Product Images from "Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription"

Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

Journal: Nucleic Acids Research

doi: 10.1093/nar/gni064

Agarose gel electrophoresis of RDA products from RNA extracted from bovine parainfluenza virus 3-infected cells. Double-stranded cDNA was synthesized from RNA of bovine parainfluenza virus 3-infected MDBK cells and subjected to RDA. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide. RDA product from the uninfected control cells was used as a negative control.
Figure Legend Snippet: Agarose gel electrophoresis of RDA products from RNA extracted from bovine parainfluenza virus 3-infected cells. Double-stranded cDNA was synthesized from RNA of bovine parainfluenza virus 3-infected MDBK cells and subjected to RDA. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide. RDA product from the uninfected control cells was used as a negative control.

Techniques Used: Agarose Gel Electrophoresis, Infection, Synthesized, Amplification, Staining, Negative Control

Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.
Figure Legend Snippet: Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.

Techniques Used: Synthesized, In Vitro, Plasmid Preparation, Agarose Gel Electrophoresis

36) Product Images from "Synthetic single domain antibodies for the conformational trapping of membrane proteins"

Article Title: Synthetic single domain antibodies for the conformational trapping of membrane proteins

Journal: eLife

doi: 10.7554/eLife.34317

FX cloning vector series for phage display and purification of sybodies and nanobodies. Sybody pools from ribosome display (or nanobodies from immunized camelids) are amplified with primers containing restriction sites of Type IIS enzyme BspQI (isoschizomer of SapI) to generate AGT and GCA overhangs. BspQI restriction sites generating the same overhangs were introduced into the backbones of vector pDX_init for phage display and pSb_init for periplasmatic expression and attachment of Myc- and His-tag. Note that in pDX_init and pSb_init the BspQI restriction sites are part of the sybody open reading frame. Finally, sybodies/nanobodies are sub-cloned from pSb_init to the destiny vectors pBXNPH3 or pBXNPHM3 for periplasmic expression. Tag-less sybodies/nanobodies for structural biology purposes can be obtained by 3C protease cleavage. Importantly, the vector series permits for PCR-free subcloning once the sybodies have been inserted into phage display vector pDX_init. The vectors were made available through Addgene (for Addgene IDs, see Table 3 ).
Figure Legend Snippet: FX cloning vector series for phage display and purification of sybodies and nanobodies. Sybody pools from ribosome display (or nanobodies from immunized camelids) are amplified with primers containing restriction sites of Type IIS enzyme BspQI (isoschizomer of SapI) to generate AGT and GCA overhangs. BspQI restriction sites generating the same overhangs were introduced into the backbones of vector pDX_init for phage display and pSb_init for periplasmatic expression and attachment of Myc- and His-tag. Note that in pDX_init and pSb_init the BspQI restriction sites are part of the sybody open reading frame. Finally, sybodies/nanobodies are sub-cloned from pSb_init to the destiny vectors pBXNPH3 or pBXNPHM3 for periplasmic expression. Tag-less sybodies/nanobodies for structural biology purposes can be obtained by 3C protease cleavage. Importantly, the vector series permits for PCR-free subcloning once the sybodies have been inserted into phage display vector pDX_init. The vectors were made available through Addgene (for Addgene IDs, see Table 3 ).

Techniques Used: Clone Assay, Plasmid Preparation, Purification, Amplification, Expressing, Polymerase Chain Reaction, Subcloning

37) Product Images from "Long non-coding RNA regulation of spermatogenesis and endosomal processes via the spectrin cytoskeleton in Drosophila"

Article Title: Long non-coding RNA regulation of spermatogenesis and endosomal processes via the spectrin cytoskeleton in Drosophila

Journal: bioRxiv

doi: 10.1101/2020.07.24.220640

The lncRNA CR45362 biochemically interacts with cytoskeletal and endosomal related proteins (A) Map of ChIRP probe locations relative to CR45362 gene. Probes 5-8 map to the deleted segment of the gene in the KO. (Image: SnapGene 4.3.4) (B) Molecular Function Gene Ontology for top ChIRP-MS proteins indicates cytoskeletal proteins bind CR45362. (C) Driver test indicates Ptc-Gal4 x UAS-GFP expresses GFP in the same region of the basal testis as is indicated by CR45362 FISH (see also Fig. 2A). (D) RNAi screen of top ChIRP-MS candidate proteins. Five pairs of testis from 3-5 day old males from each UAS-Gal4 cross were dissected of which we scored 3. If defects were seen, we repeated this screening for a second round of crosses. If both testis of a pair had the same phenotype, they were scored according to the following criteria: normal nuclear bundling, one or more scattered nuclear bundles, no nuclear bundles present, one or more individualization complexes (actin) scattered, no actin cones present, or testis deformed. (E) Results from ChIRP-MS. Columns list the number of hits (spectra) for each protein. Numbers are internally relative to each individual sample, and are used for ranking proteins, cut-off selection, and identifying background proteins. Probes 5-8 (Fig. 4A, S3) cover the CRISPR excised region, when mixed with KO samples these probes serve to identify background proteins pulled down by beads, general biotin probes, and as a negative control for the pulldown of CR45362 (Fig. S4). Each individual probe could have probe-specific background, therefore established protocols54 recommend the optimal control for a ChIRP assay is to pool probes into “Even or Odd” groups in WT samples, allowing further identification of background proteins and can be compared with the WT sample mixed with “All” probes. (F) The Ptc-Gal4 x α-Spec RNAi line has dispersed actin cones (indicated by phalloidin staining) and spermatid nuclei (yellow arrows), which phenocopied the nuclear dispersion seen in CR45362KO (G). *All scale bars represent 10 μm.
Figure Legend Snippet: The lncRNA CR45362 biochemically interacts with cytoskeletal and endosomal related proteins (A) Map of ChIRP probe locations relative to CR45362 gene. Probes 5-8 map to the deleted segment of the gene in the KO. (Image: SnapGene 4.3.4) (B) Molecular Function Gene Ontology for top ChIRP-MS proteins indicates cytoskeletal proteins bind CR45362. (C) Driver test indicates Ptc-Gal4 x UAS-GFP expresses GFP in the same region of the basal testis as is indicated by CR45362 FISH (see also Fig. 2A). (D) RNAi screen of top ChIRP-MS candidate proteins. Five pairs of testis from 3-5 day old males from each UAS-Gal4 cross were dissected of which we scored 3. If defects were seen, we repeated this screening for a second round of crosses. If both testis of a pair had the same phenotype, they were scored according to the following criteria: normal nuclear bundling, one or more scattered nuclear bundles, no nuclear bundles present, one or more individualization complexes (actin) scattered, no actin cones present, or testis deformed. (E) Results from ChIRP-MS. Columns list the number of hits (spectra) for each protein. Numbers are internally relative to each individual sample, and are used for ranking proteins, cut-off selection, and identifying background proteins. Probes 5-8 (Fig. 4A, S3) cover the CRISPR excised region, when mixed with KO samples these probes serve to identify background proteins pulled down by beads, general biotin probes, and as a negative control for the pulldown of CR45362 (Fig. S4). Each individual probe could have probe-specific background, therefore established protocols54 recommend the optimal control for a ChIRP assay is to pool probes into “Even or Odd” groups in WT samples, allowing further identification of background proteins and can be compared with the WT sample mixed with “All” probes. (F) The Ptc-Gal4 x α-Spec RNAi line has dispersed actin cones (indicated by phalloidin staining) and spermatid nuclei (yellow arrows), which phenocopied the nuclear dispersion seen in CR45362KO (G). *All scale bars represent 10 μm.

Techniques Used: Fluorescence In Situ Hybridization, Selection, CRISPR, Negative Control, Staining

The lncRNA CR45362 has positive effect on endo/lysosomal activity and fertility A) Diagram of CR45362, SNP’s identified in our lysosomal GWAS, primers, and CRISPR gRNAs. (Image: SnapGene 4.3.4) (B) DNA gel using primers flanking deletion target segment (p473 474). WT has band at 2182 demonstrating the primer targeted segment has no deletion, heterozygous CR45362 KO (+/-) has the 2182bp band but also the expected 508bp band for the deletion on the homologous chromosome, and KO has only the 508bp band indicating successful deletion in the CR45362 gene. The 508bp band was excised for sequencing and alignment (Fig. S1). (C) DNA gel of cDNA converted from CR45362 RNA using primers 1421 and 1423 (as diagrammed in 1A) that precisely correspond to the predicted ends of the lncRNA. The predicted size of CR45362 if spliced as annotated is 1207bp while unspliced is 1338bp. The gel revealed only a single band near the 1200bp mark indicating that there is a single isoform in our WT line and it is consistent with predicted splicing. Full length gel can be found in Fig. S1. (D,E) Lysotracker staining of WT and CR45362KO fed and fasted fat bodies. Knockout of the lncRNA CR45362 results in lowered Lysotracker staining under both conditions. One replicate consists of the average of three circular areas of 100μm per fly fat body measured by Cell Sens software for percentage of measured area containing fluorescence. From left to right n=7,12,6,7,6,8. Fed/fasting interaction calculated using Two-way Anova column factor (** p = 0.0034), WT vs KO fed two tailed unpaired t-test (*** p = 0.001) mean+/-s.e.m. (F) Fertility was tested by crossing individual CR45362KO male flies with WT, KO, or heterozygotes for the KO virgin females. These crosses were unable to produce offspring. However, KO and heterozygous females produced adult progeny and were fully fertile, indicating a male homozygous KO sterility defect. From top to bottom n=4,13,27,3,2,5,3,6. *All scale bars represent 10 μm. *For all figures: WT = (w1118), +/-= Heterozygous, KO = CR45362 mutant.
Figure Legend Snippet: The lncRNA CR45362 has positive effect on endo/lysosomal activity and fertility A) Diagram of CR45362, SNP’s identified in our lysosomal GWAS, primers, and CRISPR gRNAs. (Image: SnapGene 4.3.4) (B) DNA gel using primers flanking deletion target segment (p473 474). WT has band at 2182 demonstrating the primer targeted segment has no deletion, heterozygous CR45362 KO (+/-) has the 2182bp band but also the expected 508bp band for the deletion on the homologous chromosome, and KO has only the 508bp band indicating successful deletion in the CR45362 gene. The 508bp band was excised for sequencing and alignment (Fig. S1). (C) DNA gel of cDNA converted from CR45362 RNA using primers 1421 and 1423 (as diagrammed in 1A) that precisely correspond to the predicted ends of the lncRNA. The predicted size of CR45362 if spliced as annotated is 1207bp while unspliced is 1338bp. The gel revealed only a single band near the 1200bp mark indicating that there is a single isoform in our WT line and it is consistent with predicted splicing. Full length gel can be found in Fig. S1. (D,E) Lysotracker staining of WT and CR45362KO fed and fasted fat bodies. Knockout of the lncRNA CR45362 results in lowered Lysotracker staining under both conditions. One replicate consists of the average of three circular areas of 100μm per fly fat body measured by Cell Sens software for percentage of measured area containing fluorescence. From left to right n=7,12,6,7,6,8. Fed/fasting interaction calculated using Two-way Anova column factor (** p = 0.0034), WT vs KO fed two tailed unpaired t-test (*** p = 0.001) mean+/-s.e.m. (F) Fertility was tested by crossing individual CR45362KO male flies with WT, KO, or heterozygotes for the KO virgin females. These crosses were unable to produce offspring. However, KO and heterozygous females produced adult progeny and were fully fertile, indicating a male homozygous KO sterility defect. From top to bottom n=4,13,27,3,2,5,3,6. *All scale bars represent 10 μm. *For all figures: WT = (w1118), +/-= Heterozygous, KO = CR45362 mutant.

Techniques Used: Activity Assay, GWAS, CRISPR, Sequencing, Staining, Knock-Out, Software, Fluorescence, Two Tailed Test, Produced, Sterility, Mutagenesis

38) Product Images from "Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription"

Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

Journal: Nucleic Acids Research

doi: 10.1093/nar/gni064

Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.
Figure Legend Snippet: Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.

Techniques Used: Synthesized, In Vitro, Plasmid Preparation, Agarose Gel Electrophoresis

39) Product Images from "Cloning and characterization of MAP2191 gene, a mammalian cell entry antigen of Mycobacterium avium subspecies paratuberculosis"

Article Title: Cloning and characterization of MAP2191 gene, a mammalian cell entry antigen of Mycobacterium avium subspecies paratuberculosis

Journal: Molecular Biology Research Communications

doi: 10.22099/mbrc.2018.30979.1354

(A) Restriction digestion of confirmed colony PCR positive pZ57R/T mce -whole clones: lane M: 1kb DNA ladder (#SM0313, Fermentas), lanes 1, 2 and 3: Positive clones of PTZ57R/T mce -whole plasmid digested with Bam HI and Hin dIII enzymes; Lanes 5, 6 and 7: Plasmid isolated of positive PTZ57R/T mce -whole clones. (B) Restriction Digestion of confirmed colony PCR positive pET28a mce -whole clones; lane M: 1kb DNA ladder (#SM0313, Fermentas),lanes 2 and 4: Positive clones of pET28a mce -whole plasmid.
Figure Legend Snippet: (A) Restriction digestion of confirmed colony PCR positive pZ57R/T mce -whole clones: lane M: 1kb DNA ladder (#SM0313, Fermentas), lanes 1, 2 and 3: Positive clones of PTZ57R/T mce -whole plasmid digested with Bam HI and Hin dIII enzymes; Lanes 5, 6 and 7: Plasmid isolated of positive PTZ57R/T mce -whole clones. (B) Restriction Digestion of confirmed colony PCR positive pET28a mce -whole clones; lane M: 1kb DNA ladder (#SM0313, Fermentas),lanes 2 and 4: Positive clones of pET28a mce -whole plasmid.

Techniques Used: Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Isolation

40) Product Images from "Cloning and characterization of MAP2191 gene, a mammalian cell entry antigen of Mycobacterium avium subspecies paratuberculosis"

Article Title: Cloning and characterization of MAP2191 gene, a mammalian cell entry antigen of Mycobacterium avium subspecies paratuberculosis

Journal: Molecular Biology Research Communications

doi: 10.22099/mbrc.2018.30979.1354

(A) Restriction digestion of confirmed colony PCR positive pZ57R/T mce -whole clones: lane M: 1kb DNA ladder (#SM0313, Fermentas), lanes 1, 2 and 3: Positive clones of PTZ57R/T mce -whole plasmid digested with Bam HI and Hin dIII enzymes; Lanes 5, 6 and 7: Plasmid isolated of positive PTZ57R/T mce -whole clones. (B) Restriction Digestion of confirmed colony PCR positive pET28a mce -whole clones; lane M: 1kb DNA ladder (#SM0313, Fermentas),lanes 2 and 4: Positive clones of pET28a mce -whole plasmid.
Figure Legend Snippet: (A) Restriction digestion of confirmed colony PCR positive pZ57R/T mce -whole clones: lane M: 1kb DNA ladder (#SM0313, Fermentas), lanes 1, 2 and 3: Positive clones of PTZ57R/T mce -whole plasmid digested with Bam HI and Hin dIII enzymes; Lanes 5, 6 and 7: Plasmid isolated of positive PTZ57R/T mce -whole clones. (B) Restriction Digestion of confirmed colony PCR positive pET28a mce -whole clones; lane M: 1kb DNA ladder (#SM0313, Fermentas),lanes 2 and 4: Positive clones of pET28a mce -whole plasmid.

Techniques Used: Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Isolation

(A) Analysis of Mce-whole Protein expressed by SDS PAGE. (Left to Right), lane M: Protein molecular weight marker (SL7012, CinnaGen), lane 1: pET28a.Mce.whole Elution 1, lane 2: pET28a.Mce.whole Supernatant, lane 3: pET28a.Mce.whole Pellet, lane 4. pET28a.Mce.whole Crude; (B) Western blot analysis (from left to right) lane M: Protein molecular weight marker (SL7012, CinnaGen), lane 1: pET28a.Mce.whole Elution 1, lane 2: p1ET28a.Mce.whole Supernatant, lane 3: pET28a.Mce.whole Pellet, lane 4. pET28a.Mce.whole Crude.
Figure Legend Snippet: (A) Analysis of Mce-whole Protein expressed by SDS PAGE. (Left to Right), lane M: Protein molecular weight marker (SL7012, CinnaGen), lane 1: pET28a.Mce.whole Elution 1, lane 2: pET28a.Mce.whole Supernatant, lane 3: pET28a.Mce.whole Pellet, lane 4. pET28a.Mce.whole Crude; (B) Western blot analysis (from left to right) lane M: Protein molecular weight marker (SL7012, CinnaGen), lane 1: pET28a.Mce.whole Elution 1, lane 2: p1ET28a.Mce.whole Supernatant, lane 3: pET28a.Mce.whole Pellet, lane 4. pET28a.Mce.whole Crude.

Techniques Used: SDS Page, Molecular Weight, Marker, Western Blot

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Agarose Gel Electrophoresis:

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Article Snippet: .. The digestion products were analyzed by agarose gel electrophoresis (stained with GelRel) with the marker pUC19 DNA/MspI (Hpall) (Thermo Scientific). .. For positive controls, HEK293 were transfected with pRK5-GluR1i-L497Y-(Ser831-Ser845), pRetroCAG-GluA2(Q583R), pRK5-GluA3 flip, or pCI-EGFPGluA4 using lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

Concentration Assay:

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Article Snippet: .. DNA Relaxation Reaction mixtures containing 0.4 mg pUC19 plasmid DNA (MBI Fermentas, Vilnius, Lithuania) and 2.5 μg nuclear extracts (NE) from 8-Cl-Ado-exposed or -unexposed cells, or 5 mM 3-aminobenzamide (PARP inhibitor) in 20 μL relaxation buffer (50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 5 mM MgCl2 ) were incubated at 37 °C for 30 min and stopped by adding sodium dodecyl sulphate (SDS) and ethylenediaminetetraacetic acid (EDTA) to a final concentration of 0.1% and 10 mM, respectively. .. DNA was ethanol precipitated, and subjected to electrophoresis in 1% agarose gels.

Incubation:

Article Title: DNA Damage-Response Pathway Heterogeneity of Human Lung Cancer A549 and H1299 Cells Determines Sensitivity to 8-Chloro-Adenosine
Article Snippet: .. DNA Relaxation Reaction mixtures containing 0.4 mg pUC19 plasmid DNA (MBI Fermentas, Vilnius, Lithuania) and 2.5 μg nuclear extracts (NE) from 8-Cl-Ado-exposed or -unexposed cells, or 5 mM 3-aminobenzamide (PARP inhibitor) in 20 μL relaxation buffer (50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 5 mM MgCl2 ) were incubated at 37 °C for 30 min and stopped by adding sodium dodecyl sulphate (SDS) and ethylenediaminetetraacetic acid (EDTA) to a final concentration of 0.1% and 10 mM, respectively. .. DNA was ethanol precipitated, and subjected to electrophoresis in 1% agarose gels.

Marker:

Article Title: Glutamate Activates AMPA Receptor Conductance in the Developing Schwann Cells of the Mammalian Peripheral Nerves
Article Snippet: .. The digestion products were analyzed by agarose gel electrophoresis (stained with GelRel) with the marker pUC19 DNA/MspI (Hpall) (Thermo Scientific). .. For positive controls, HEK293 were transfected with pRK5-GluR1i-L497Y-(Ser831-Ser845), pRetroCAG-GluA2(Q583R), pRK5-GluA3 flip, or pCI-EGFPGluA4 using lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

Staining:

Article Title: Glutamate Activates AMPA Receptor Conductance in the Developing Schwann Cells of the Mammalian Peripheral Nerves
Article Snippet: .. The digestion products were analyzed by agarose gel electrophoresis (stained with GelRel) with the marker pUC19 DNA/MspI (Hpall) (Thermo Scientific). .. For positive controls, HEK293 were transfected with pRK5-GluR1i-L497Y-(Ser831-Ser845), pRetroCAG-GluA2(Q583R), pRK5-GluA3 flip, or pCI-EGFPGluA4 using lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

Plasmid Preparation:

Article Title: The structural characterization of a prophage-encoded extracellular DNase from Streptococcus pyogenes
Article Snippet: .. Preparation of DNA substrate The plasmid vector pUC19 was linearized with EcoRI (Fermentas) and used as a homogeneous substrate for the continuous assay. .. The linearization was verified by agarose gel electrophoresis, and the restriction endonuclease inactivated in accordance with the manufacturer's instructions (heat denaturation at 65°C for 20 min).

Article Title: An artificial metalloenzyme for catalytic cancer-specific DNA cleavage and operando imaging
Article Snippet: .. The Supercoiled pUC19 plasmid DNA with 2686 bp was purchased from Thermo Fisher Scientific for the DNA double-strand break assays. .. Cell culture 1640 medium and fetal bovine serum (FBS) were purchased from HyClone.

Article Title: DNA Damage-Response Pathway Heterogeneity of Human Lung Cancer A549 and H1299 Cells Determines Sensitivity to 8-Chloro-Adenosine
Article Snippet: .. DNA Relaxation Reaction mixtures containing 0.4 mg pUC19 plasmid DNA (MBI Fermentas, Vilnius, Lithuania) and 2.5 μg nuclear extracts (NE) from 8-Cl-Ado-exposed or -unexposed cells, or 5 mM 3-aminobenzamide (PARP inhibitor) in 20 μL relaxation buffer (50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 5 mM MgCl2 ) were incubated at 37 °C for 30 min and stopped by adding sodium dodecyl sulphate (SDS) and ethylenediaminetetraacetic acid (EDTA) to a final concentration of 0.1% and 10 mM, respectively. .. DNA was ethanol precipitated, and subjected to electrophoresis in 1% agarose gels.

Article Title: Evaluation of digital real-time PCR assay as a molecular diagnostic tool for single-cell analysis
Article Snippet: .. The pUC19 plasmid DNA standard was quantitated using Nanodrop™ (Thermo Fisher Scientific, Taiwan) and converted into the DNA copy number used in the calculations in this study. ..

Article Title: Mycoplasma fermentans Inhibits the Activity of Cellular DNA Topoisomerase I by Activation of PARP1 and Alters the Efficacy of Its Anti-Cancer Inhibitor
Article Snippet: .. Supercoiled DNA plasmid pUC19 was purchased from MBI Fermentas (Hanover, MD, USA). .. PD98059 and 3-aminobenzamide (3AB) were purchased from Sigma-Aldrich (Rehovot, Israel).

Article Title: The Post-Synaptic Function of Brca2
Article Snippet: .. DNA Unwinding Assay Relaxed DNA was prepared in a 50 µl reaction mixture by incubating negatively supercoiled plasmid pUC19 DNA (0.5 µg) with calf thymus Topoisomerase I (1 Unit; Invitrogen) in buffer containing 50 mM Tris HCl pH 7.5, 50 mM KCl, 10 mM MgCl2 , 0.5 mM DTT, 0.1 mM EDTA, 100 µg ml−1 BSA for 30 min at 37 °C. .. To test the unwinding activity of RAD51, RAD51 K133A, RAD51 K133R, these proteins were incubated with relaxed pUC19 (25 µM, nt) and unlabeled #160 (12.5 µM, nt) in the D-loop formation buffer supplemented with 10 mM magnesium acetate for 5 min at 37 °C.

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