Structured Review

TaKaRa dna manipulation
ACSL3 but not <t>ACSL6</t> is recruited into the Ct inclusion. HeLa cells were grown on coverslips and infected with Ct strain D at a MOI of 1 in cell culture medium without antibiotics and cycloheximide. Cells were fixed 36 hr post- infection. <t>DNA</t> was detected with Hoechst 33258 dye (blue). Bacteria were detected with a rabbit antibody against IncA, which was stained with an anti-rabbit antibody labeled with AlexaFluor®488 revealing the inclusion membrane (green, panels A to C) or with a mouse antibody against LPS, which was stained with Cy TM 3-conjugated anti-mouse antibody (red, panel D). Long-chain acyl-CoA synthetase 3, ACSL3, was detected with a mouse monoclonal antibody (red, panel B and C) and ACSL6 protein was detected with a rabbit antibody (green, panel D). The inclusion membrane of one representative inclusion is indicated with an arrow in panel A and B. Panel C displays a cropped portion of the image shown in panel B. Insets in panel C and D display the orthogonal (z, x) and (z, y) views of the deconvolved Z stack merged images. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in panels and insets represent 10 µm. Note that the two insets of panel C were slightly scaled up compare to the main image. Nuclei and inclusion are indicated with N and Inc, respectively. ACSL3 protein was detected inside the inclusion (panel B and C) and ACSL6 was not (panel D).
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Images

1) Product Images from "Eukaryotic Protein Recruitment into the Chlamydia Inclusion: Implications for Survival and Growth"

Article Title: Eukaryotic Protein Recruitment into the Chlamydia Inclusion: Implications for Survival and Growth

Journal: PLoS ONE

doi: 10.1371/journal.pone.0036843

ACSL3 but not ACSL6 is recruited into the Ct inclusion. HeLa cells were grown on coverslips and infected with Ct strain D at a MOI of 1 in cell culture medium without antibiotics and cycloheximide. Cells were fixed 36 hr post- infection. DNA was detected with Hoechst 33258 dye (blue). Bacteria were detected with a rabbit antibody against IncA, which was stained with an anti-rabbit antibody labeled with AlexaFluor®488 revealing the inclusion membrane (green, panels A to C) or with a mouse antibody against LPS, which was stained with Cy TM 3-conjugated anti-mouse antibody (red, panel D). Long-chain acyl-CoA synthetase 3, ACSL3, was detected with a mouse monoclonal antibody (red, panel B and C) and ACSL6 protein was detected with a rabbit antibody (green, panel D). The inclusion membrane of one representative inclusion is indicated with an arrow in panel A and B. Panel C displays a cropped portion of the image shown in panel B. Insets in panel C and D display the orthogonal (z, x) and (z, y) views of the deconvolved Z stack merged images. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in panels and insets represent 10 µm. Note that the two insets of panel C were slightly scaled up compare to the main image. Nuclei and inclusion are indicated with N and Inc, respectively. ACSL3 protein was detected inside the inclusion (panel B and C) and ACSL6 was not (panel D).
Figure Legend Snippet: ACSL3 but not ACSL6 is recruited into the Ct inclusion. HeLa cells were grown on coverslips and infected with Ct strain D at a MOI of 1 in cell culture medium without antibiotics and cycloheximide. Cells were fixed 36 hr post- infection. DNA was detected with Hoechst 33258 dye (blue). Bacteria were detected with a rabbit antibody against IncA, which was stained with an anti-rabbit antibody labeled with AlexaFluor®488 revealing the inclusion membrane (green, panels A to C) or with a mouse antibody against LPS, which was stained with Cy TM 3-conjugated anti-mouse antibody (red, panel D). Long-chain acyl-CoA synthetase 3, ACSL3, was detected with a mouse monoclonal antibody (red, panel B and C) and ACSL6 protein was detected with a rabbit antibody (green, panel D). The inclusion membrane of one representative inclusion is indicated with an arrow in panel A and B. Panel C displays a cropped portion of the image shown in panel B. Insets in panel C and D display the orthogonal (z, x) and (z, y) views of the deconvolved Z stack merged images. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in panels and insets represent 10 µm. Note that the two insets of panel C were slightly scaled up compare to the main image. Nuclei and inclusion are indicated with N and Inc, respectively. ACSL3 protein was detected inside the inclusion (panel B and C) and ACSL6 was not (panel D).

Techniques Used: Infection, Cell Culture, Staining, Labeling, Microscopy

LPCAT1 enzyme is tightly associated with the inclusion membrane. HeLa cells were grown on coverslips in cell culture medium without antibiotics and cycloheximide, and were transfected with a DNA construct expressing a GFP protein fused to acyl-CoA:lysophosphatidylcholine acyltransferase 1, LPCAT1. After 24 hr, cells were infected with Ct strain D at a MOI of 1 and were fixed 44 hr post-infection. DNA was detected with the Hoechst dye (blue) and the bacteria were detected with a rabbit antibody against IncA revealing the inclusion membrane (red). A deconvolved Z stack merged image with the GFP signal is shown in panel A. A cropped section of the image is displayed in the inset. White arrowheads indicate the position of the inclusion membrane. The tight association of LPCAT1with the inclusion membrane and overlap of the IncA and GFP signals, detected as yellow dots, can be observed in the inset. Panel B shows traces of the intensity of the signal for DNA, IncA and LPCAT1 (y axis) plotted in function of the distance (x axis) from area I to II as indicated by a red arrow on panel A. GFP and IncA signals co-localized in area I but not in area II. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in the panel and the inset represent 10 and 1 µm, respectively.
Figure Legend Snippet: LPCAT1 enzyme is tightly associated with the inclusion membrane. HeLa cells were grown on coverslips in cell culture medium without antibiotics and cycloheximide, and were transfected with a DNA construct expressing a GFP protein fused to acyl-CoA:lysophosphatidylcholine acyltransferase 1, LPCAT1. After 24 hr, cells were infected with Ct strain D at a MOI of 1 and were fixed 44 hr post-infection. DNA was detected with the Hoechst dye (blue) and the bacteria were detected with a rabbit antibody against IncA revealing the inclusion membrane (red). A deconvolved Z stack merged image with the GFP signal is shown in panel A. A cropped section of the image is displayed in the inset. White arrowheads indicate the position of the inclusion membrane. The tight association of LPCAT1with the inclusion membrane and overlap of the IncA and GFP signals, detected as yellow dots, can be observed in the inset. Panel B shows traces of the intensity of the signal for DNA, IncA and LPCAT1 (y axis) plotted in function of the distance (x axis) from area I to II as indicated by a red arrow on panel A. GFP and IncA signals co-localized in area I but not in area II. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in the panel and the inset represent 10 and 1 µm, respectively.

Techniques Used: Cell Culture, Transfection, Construct, Expressing, Infection, Microscopy

2) Product Images from "Partially Overlapping Primer-Based PCR for Genome Walking"

Article Title: Partially Overlapping Primer-Based PCR for Genome Walking

Journal: PLoS ONE

doi: 10.1371/journal.pone.0120139

Chromosome walking of the gadA locus of Lactobacillus brevis NCL912 (a), human aldolase A gene (b), malQ of Pichia pastoris GS115 (c), and hyg of rice (d). I: walking into 5’ regions of the genes (locus); II: walking into 3’ regions of the genes (locus). Each walking experiment contained four sets of PCRs that respectively utilized the four POP primer sets, POP1, POP2, POP3, and POP4, paired with a specific primer set. For each set of PCRs, only the results of secondary PCR (left lane) and tertiary (right lane) PCR are presented. White arrows indicate target bands. M1: DL2000 DNA marker. M2: λ-Hind III digest DNA Marker. M3: DL5000 DNA marker.
Figure Legend Snippet: Chromosome walking of the gadA locus of Lactobacillus brevis NCL912 (a), human aldolase A gene (b), malQ of Pichia pastoris GS115 (c), and hyg of rice (d). I: walking into 5’ regions of the genes (locus); II: walking into 3’ regions of the genes (locus). Each walking experiment contained four sets of PCRs that respectively utilized the four POP primer sets, POP1, POP2, POP3, and POP4, paired with a specific primer set. For each set of PCRs, only the results of secondary PCR (left lane) and tertiary (right lane) PCR are presented. White arrows indicate target bands. M1: DL2000 DNA marker. M2: λ-Hind III digest DNA Marker. M3: DL5000 DNA marker.

Techniques Used: Chromosome Walking, Polymerase Chain Reaction, Marker

Overview of primer partially overlapping-based PCR. The first five high-stringency cycles (HSC) of each PCR are to increase copies of the single-stranded DNA of interest. The one low-stringency cycle (LSC) of primary PCR facilitates POP-P annealing to the target DNA and extension towards SP1. The one reduced-stringency cycle (RSC) of secondary PCR allowed POP-S to bind to the POP-P annealing site. A double-stranded target molecule was synthesized in the first HSC following LSC/RSC, and served as the template for the remaining HSCs; non-specific amplification was inhibited because the double-stranded form could not be obtained from a non-target single strand. Solid lines: the known sequence; dotted lines: the unknown sequence; thick black arrows with different heads: nested, specific primers; hollow arrows with different tails: POP primers; gray arrows: primers complementary sequences.
Figure Legend Snippet: Overview of primer partially overlapping-based PCR. The first five high-stringency cycles (HSC) of each PCR are to increase copies of the single-stranded DNA of interest. The one low-stringency cycle (LSC) of primary PCR facilitates POP-P annealing to the target DNA and extension towards SP1. The one reduced-stringency cycle (RSC) of secondary PCR allowed POP-S to bind to the POP-P annealing site. A double-stranded target molecule was synthesized in the first HSC following LSC/RSC, and served as the template for the remaining HSCs; non-specific amplification was inhibited because the double-stranded form could not be obtained from a non-target single strand. Solid lines: the known sequence; dotted lines: the unknown sequence; thick black arrows with different heads: nested, specific primers; hollow arrows with different tails: POP primers; gray arrows: primers complementary sequences.

Techniques Used: Polymerase Chain Reaction, Synthesized, Amplification, Sequencing

3) Product Images from "Stepwise partially overlapping primer-based PCR for genome walking"

Article Title: Stepwise partially overlapping primer-based PCR for genome walking

Journal: AMB Express

doi: 10.1186/s13568-018-0610-7

Genome walking of the gadA locus of Lactobacillus brevis NCL912 and hyg of rice. Each walking experiment contained four sets of PCR reactions that respectively utilized the four SWPOP primer sets, SWPOP1 (I), SWPOP2 (II), SWPOP3 (III) and SWPOP4 (IV), paired with a specific primer set. For each set of PCR reactions, the results of primary PCR (P), secondary PCR (S) and tertiary PCR (T) are presented. White arrows indicate target bands. M: DL2000 DNA marker
Figure Legend Snippet: Genome walking of the gadA locus of Lactobacillus brevis NCL912 and hyg of rice. Each walking experiment contained four sets of PCR reactions that respectively utilized the four SWPOP primer sets, SWPOP1 (I), SWPOP2 (II), SWPOP3 (III) and SWPOP4 (IV), paired with a specific primer set. For each set of PCR reactions, the results of primary PCR (P), secondary PCR (S) and tertiary PCR (T) are presented. White arrows indicate target bands. M: DL2000 DNA marker

Techniques Used: Polymerase Chain Reaction, Marker

Outline of stepwise partially overlapping primer-based PCR. The first five high-stringency cycles (HSCs) of each PCR were carried out to increase the copy number of specific single-stranded DNA (ssDNA) of interest with different length. The one low-stringency cycle (LSC) of primary PCR allowed SWPOP-P to anneal to the target DNA and extend towards SP-P. The one reduced-stringency cycle (RSC) of secondary/tertiary PCR allowed SWPOP-S/SWPOP-T to bind to the prior SWPOP complementary site(s). A double-stranded target DNA molecule was obtained in the first HSC following LSC/RSC, and served as the template for the remaining twenty-four HSCs. Non-target amplification was suppressed because the double-stranded form could not be synthesized from a non-target single strand. Drawings on the right side: potential nonspecific (non-target) amplifications; SP-P, SP-S, and SP-T: specific primer for primary, secondary, and tertiary PCR, respectively; SWPOP-P, SWPOP-S, and SWPOP-T: stepwise partially overlapping primer for primary, secondary, and tertiary PCR, respectively; solid lines: the known sequence; dotted lines: the unknown sequence; grey arrows: primers complementary sites
Figure Legend Snippet: Outline of stepwise partially overlapping primer-based PCR. The first five high-stringency cycles (HSCs) of each PCR were carried out to increase the copy number of specific single-stranded DNA (ssDNA) of interest with different length. The one low-stringency cycle (LSC) of primary PCR allowed SWPOP-P to anneal to the target DNA and extend towards SP-P. The one reduced-stringency cycle (RSC) of secondary/tertiary PCR allowed SWPOP-S/SWPOP-T to bind to the prior SWPOP complementary site(s). A double-stranded target DNA molecule was obtained in the first HSC following LSC/RSC, and served as the template for the remaining twenty-four HSCs. Non-target amplification was suppressed because the double-stranded form could not be synthesized from a non-target single strand. Drawings on the right side: potential nonspecific (non-target) amplifications; SP-P, SP-S, and SP-T: specific primer for primary, secondary, and tertiary PCR, respectively; SWPOP-P, SWPOP-S, and SWPOP-T: stepwise partially overlapping primer for primary, secondary, and tertiary PCR, respectively; solid lines: the known sequence; dotted lines: the unknown sequence; grey arrows: primers complementary sites

Techniques Used: Polymerase Chain Reaction, Amplification, Synthesized, Sequencing

4) Product Images from "Mutations Affecting DNA-Binding Activity of the MexR Repressor of mexR-mexA-mexB-oprM Operon Expression"

Article Title: Mutations Affecting DNA-Binding Activity of the MexR Repressor of mexR-mexA-mexB-oprM Operon Expression

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.20.6195-6198.2003

Properties of the mutant MexR-His 6 protein. (A) Expression of the MexR protein. PAO4290 cells harboring pMMB67HE with or without mexR-his 6 were grown in the presence of 1 mM IPTG. Cell lysate was prepared by disintegrating cells in a solution of 10 mM Tris-2% Triton X-100-1 mM MgCl 2 (pH 7.9) with ultrasonic oscillation and then centrifuging at 100,000 × g ). Preparation of the mexOP ). Lanes: 1, pMMB67HE without mexR ; 2, pMMB67HE encoding wild-type MexR-His 6 ; 3, Leu45Pro; 4, Ile46Asn; 5, Leu57Pro; 6, Leu57Arg; 7, Thr69Ile; 8, Ile72Asn; 9, Leu75Pro; 10, Leu75Arg; 11, Arg83Cys; 12, Arg91Cys; 13, Arg91His; 14, purified MexR-His. (B) Gel retardation assays by MexR-His 6 . The mexOP probe DNA (final concentration, 10 nM) was incubated with homogeneously purified MexR-His 6 derived from E. coli DH5α (final concentration, 0.2 μM) in a solution of 4 mM Tris-4 mM HEPES-25 mM KCl-1 mM EDTA-25% glycerol (pH 7.9), and the 7.5-μl mixture was subjected to electrophoresis in 4% polyacrylamide gels (in 0.25× Tris-borate-EDTA). The gel was soaked in 10,000-fold-diluted CYBR Green I (BioWhittaker Molecular Applications), and DNA was visualized with UV light at 254 nm. Lanes: 1, DNA probe without MexR; 2, wild-type MexR-His; 3, Leu57Pro; 4, Leu57Arg; 5, Thr69Ile; 6, Ile72Asn; 7, Leu75Pro; 8, Leu75Arg; 9, Arg83Cys; 10, Arg91Cys; 11, Arg91His. (C) Cross-linking experiment with mutant MexR. A representative mutant MexR (Arg91His) protein, 6 μg/20 μl, was mixed with 10 μg of disuccinimydyl suberate/μl and incubated at 4°C for 30 min. To the mixture was added 10 μl of 500 mM Tris-HCl (pH 8.0) and 30 μl of solubilizer, and the mixture was heated at 100°C for 5 min. A sample containing 2 μg of MexR was applied to a 14% polyacrylamide gel. Lane 1, size markers; lane 2,Arg91His without disuccinimydyl suberate; lane 3, Arg91His with disuccinimydyl suberate. Wild-type MexR and other mutant MexR proteins showed essentially the same gel profile (data not shown). A significant amount of protein bands at the position corresponding to the dimer in the sample without disuccinimydyl suberate also appeared with wild-type MexR (not shown). Retarded mobility of disuccinimydyl suberate-treated MexR is due to bound disuccinimydyl suberates.
Figure Legend Snippet: Properties of the mutant MexR-His 6 protein. (A) Expression of the MexR protein. PAO4290 cells harboring pMMB67HE with or without mexR-his 6 were grown in the presence of 1 mM IPTG. Cell lysate was prepared by disintegrating cells in a solution of 10 mM Tris-2% Triton X-100-1 mM MgCl 2 (pH 7.9) with ultrasonic oscillation and then centrifuging at 100,000 × g ). Preparation of the mexOP ). Lanes: 1, pMMB67HE without mexR ; 2, pMMB67HE encoding wild-type MexR-His 6 ; 3, Leu45Pro; 4, Ile46Asn; 5, Leu57Pro; 6, Leu57Arg; 7, Thr69Ile; 8, Ile72Asn; 9, Leu75Pro; 10, Leu75Arg; 11, Arg83Cys; 12, Arg91Cys; 13, Arg91His; 14, purified MexR-His. (B) Gel retardation assays by MexR-His 6 . The mexOP probe DNA (final concentration, 10 nM) was incubated with homogeneously purified MexR-His 6 derived from E. coli DH5α (final concentration, 0.2 μM) in a solution of 4 mM Tris-4 mM HEPES-25 mM KCl-1 mM EDTA-25% glycerol (pH 7.9), and the 7.5-μl mixture was subjected to electrophoresis in 4% polyacrylamide gels (in 0.25× Tris-borate-EDTA). The gel was soaked in 10,000-fold-diluted CYBR Green I (BioWhittaker Molecular Applications), and DNA was visualized with UV light at 254 nm. Lanes: 1, DNA probe without MexR; 2, wild-type MexR-His; 3, Leu57Pro; 4, Leu57Arg; 5, Thr69Ile; 6, Ile72Asn; 7, Leu75Pro; 8, Leu75Arg; 9, Arg83Cys; 10, Arg91Cys; 11, Arg91His. (C) Cross-linking experiment with mutant MexR. A representative mutant MexR (Arg91His) protein, 6 μg/20 μl, was mixed with 10 μg of disuccinimydyl suberate/μl and incubated at 4°C for 30 min. To the mixture was added 10 μl of 500 mM Tris-HCl (pH 8.0) and 30 μl of solubilizer, and the mixture was heated at 100°C for 5 min. A sample containing 2 μg of MexR was applied to a 14% polyacrylamide gel. Lane 1, size markers; lane 2,Arg91His without disuccinimydyl suberate; lane 3, Arg91His with disuccinimydyl suberate. Wild-type MexR and other mutant MexR proteins showed essentially the same gel profile (data not shown). A significant amount of protein bands at the position corresponding to the dimer in the sample without disuccinimydyl suberate also appeared with wild-type MexR (not shown). Retarded mobility of disuccinimydyl suberate-treated MexR is due to bound disuccinimydyl suberates.

Techniques Used: Mutagenesis, Expressing, Purification, Electrophoretic Mobility Shift Assay, Concentration Assay, Incubation, Derivative Assay, Electrophoresis

5) Product Images from "An A-Factor-Dependent Extracytoplasmic Function Sigma Factor (?AdsA) That Is Essential for Morphological Development in Streptomyces griseus"

Article Title: An A-Factor-Dependent Extracytoplasmic Function Sigma Factor (?AdsA) That Is Essential for Morphological Development in Streptomyces griseus

Journal: Journal of Bacteriology

doi:

Gel mobility shift-PCR for isolation of DNA fragments recognized and bound by AdpA-H (A) and gel mobility shift of AdBS1 caused by AdpA-H (B). (A) The S. griseus chromosomal DNA of 300 to 500 bp obtained after Hae III digestion was sandwiched by the catch linkers, 32 P-labeled, mixed and incubated with AdpA-H, and run on a polyacrylamide gel. The amounts of AdpA-H used were 0.02 μg (lane 2), 0.2 μg (lane 3), and 1 μg (lane 4). Lane 1 is a control lane in which there was no AdpA-H. The DNA fragments retarded were recovered and subjected to a second cycle and further cycles. The mobility shift patterns after the second and third cycles are presented, showing the presence of retarded signals. The opposing arrows show the area from which DNA was extracted; the upper position was determined with a 500-bp DNA fragment, including the AdpA-binding site for strR , and the lower position was determined with a 300-bp fragment including the same AdpA-binding site, as described in Materials and Methods. (B) AdBS1 was excised by Eco RI digestion of the recombinant pUC19 plasmid, 32 P-labeled, and subjected to gel mobility shift assay. In the presence of AdpA-H (0.2 μg), AdBS1 is shifted. The positions of AdpA-H-bound (solid triangle) and free (open triangle) probes are shown.
Figure Legend Snippet: Gel mobility shift-PCR for isolation of DNA fragments recognized and bound by AdpA-H (A) and gel mobility shift of AdBS1 caused by AdpA-H (B). (A) The S. griseus chromosomal DNA of 300 to 500 bp obtained after Hae III digestion was sandwiched by the catch linkers, 32 P-labeled, mixed and incubated with AdpA-H, and run on a polyacrylamide gel. The amounts of AdpA-H used were 0.02 μg (lane 2), 0.2 μg (lane 3), and 1 μg (lane 4). Lane 1 is a control lane in which there was no AdpA-H. The DNA fragments retarded were recovered and subjected to a second cycle and further cycles. The mobility shift patterns after the second and third cycles are presented, showing the presence of retarded signals. The opposing arrows show the area from which DNA was extracted; the upper position was determined with a 500-bp DNA fragment, including the AdpA-binding site for strR , and the lower position was determined with a 300-bp fragment including the same AdpA-binding site, as described in Materials and Methods. (B) AdBS1 was excised by Eco RI digestion of the recombinant pUC19 plasmid, 32 P-labeled, and subjected to gel mobility shift assay. In the presence of AdpA-H (0.2 μg), AdBS1 is shifted. The positions of AdpA-H-bound (solid triangle) and free (open triangle) probes are shown.

Techniques Used: Mobility Shift, Polymerase Chain Reaction, Isolation, Labeling, Incubation, Binding Assay, Recombinant, Plasmid Preparation

Related Articles

DNA Extraction:

Article Title: Stepwise partially overlapping primer-based PCR for genome walking
Article Snippet: .. DNA manipulation and sequencing PCR products were purified with the MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, Dalian, China), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). .. Overview of stepwise partially overlapping primer-based PCR The outline of SWPOP-PCR is illustrated in Fig. .

Clone Assay:

Article Title: Eukaryotic Protein Recruitment into the Chlamydia Inclusion: Implications for Survival and Growth
Article Snippet: .. DNA manipulation and transfection Full-length LPCAT1 cDNA and ACSL6 cDNA , were cloned into the pAcGFP1 vector (Clontech Laboratories, Mountain View, CA) to generate a fusion of the GFP protein to their amino-terminal extremity. .. Briefly, PCR cloning was performed with High-Fidelity Expand Taq DNA polymerase (Roche Applied Science, Indianapolis, IN) using the plasmid pFK182 as template, and the following primer pair ( 5′-CTAGAGCTCAGACACAGGAGATCCTGAGGATAC-3′ ; 5′-CTAGGATCCTCACATGGAGATTGAGTAAAGCTCTTC-3′ ).

Transfection:

Article Title: Eukaryotic Protein Recruitment into the Chlamydia Inclusion: Implications for Survival and Growth
Article Snippet: .. DNA manipulation and transfection Full-length LPCAT1 cDNA and ACSL6 cDNA , were cloned into the pAcGFP1 vector (Clontech Laboratories, Mountain View, CA) to generate a fusion of the GFP protein to their amino-terminal extremity. .. Briefly, PCR cloning was performed with High-Fidelity Expand Taq DNA polymerase (Roche Applied Science, Indianapolis, IN) using the plasmid pFK182 as template, and the following primer pair ( 5′-CTAGAGCTCAGACACAGGAGATCCTGAGGATAC-3′ ; 5′-CTAGGATCCTCACATGGAGATTGAGTAAAGCTCTTC-3′ ).

Agarose Gel Electrophoresis:

Article Title: Stepwise partially overlapping primer-based PCR for genome walking
Article Snippet: .. DNA manipulation and sequencing PCR products were purified with the MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, Dalian, China), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). .. Overview of stepwise partially overlapping primer-based PCR The outline of SWPOP-PCR is illustrated in Fig. .

Article Title: Partially Overlapping Primer-Based PCR for Genome Walking
Article Snippet: .. DNA manipulation and sequencing PCR products were purified with the Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China) or ligated into pMD18-T Vector or pMD19-T Simple Vector (TaKaRa). .. Then, the recombinant plasmids were transformed into E . coli DH5α cells according to TaKaRa’s guidelines, and were sequenced by Sangon Biotech Co., Ltd.

Expressing:

Article Title: Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position
Article Snippet: .. E. coli strains JM109 for DNA manipulation and BL21-CodonPlus(DE3)-RIPL for protein expression were obtained, respectively, from TaKaRa Bio, Inc., and Stratagene (La Jolla, CA, USA). .. The plasmid vector pET-21a(+) was purchased from Novagen (Madison, WI, USA).

Purification:

Article Title: Stepwise partially overlapping primer-based PCR for genome walking
Article Snippet: .. DNA manipulation and sequencing PCR products were purified with the MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, Dalian, China), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). .. Overview of stepwise partially overlapping primer-based PCR The outline of SWPOP-PCR is illustrated in Fig. .

Article Title: Partially Overlapping Primer-Based PCR for Genome Walking
Article Snippet: .. DNA manipulation and sequencing PCR products were purified with the Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China) or ligated into pMD18-T Vector or pMD19-T Simple Vector (TaKaRa). .. Then, the recombinant plasmids were transformed into E . coli DH5α cells according to TaKaRa’s guidelines, and were sequenced by Sangon Biotech Co., Ltd.

Sequencing:

Article Title: Stepwise partially overlapping primer-based PCR for genome walking
Article Snippet: .. DNA manipulation and sequencing PCR products were purified with the MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, Dalian, China), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). .. Overview of stepwise partially overlapping primer-based PCR The outline of SWPOP-PCR is illustrated in Fig. .

Article Title: Partially Overlapping Primer-Based PCR for Genome Walking
Article Snippet: .. DNA manipulation and sequencing PCR products were purified with the Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China) or ligated into pMD18-T Vector or pMD19-T Simple Vector (TaKaRa). .. Then, the recombinant plasmids were transformed into E . coli DH5α cells according to TaKaRa’s guidelines, and were sequenced by Sangon Biotech Co., Ltd.

DNA Purification:

Article Title: Partially Overlapping Primer-Based PCR for Genome Walking
Article Snippet: .. DNA manipulation and sequencing PCR products were purified with the Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China) or ligated into pMD18-T Vector or pMD19-T Simple Vector (TaKaRa). .. Then, the recombinant plasmids were transformed into E . coli DH5α cells according to TaKaRa’s guidelines, and were sequenced by Sangon Biotech Co., Ltd.

Polymerase Chain Reaction:

Article Title: Stepwise partially overlapping primer-based PCR for genome walking
Article Snippet: .. DNA manipulation and sequencing PCR products were purified with the MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, Dalian, China), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). .. Overview of stepwise partially overlapping primer-based PCR The outline of SWPOP-PCR is illustrated in Fig. .

Article Title: Partially Overlapping Primer-Based PCR for Genome Walking
Article Snippet: .. DNA manipulation and sequencing PCR products were purified with the Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China) or ligated into pMD18-T Vector or pMD19-T Simple Vector (TaKaRa). .. Then, the recombinant plasmids were transformed into E . coli DH5α cells according to TaKaRa’s guidelines, and were sequenced by Sangon Biotech Co., Ltd.

Plasmid Preparation:

Article Title: Eukaryotic Protein Recruitment into the Chlamydia Inclusion: Implications for Survival and Growth
Article Snippet: .. DNA manipulation and transfection Full-length LPCAT1 cDNA and ACSL6 cDNA , were cloned into the pAcGFP1 vector (Clontech Laboratories, Mountain View, CA) to generate a fusion of the GFP protein to their amino-terminal extremity. .. Briefly, PCR cloning was performed with High-Fidelity Expand Taq DNA polymerase (Roche Applied Science, Indianapolis, IN) using the plasmid pFK182 as template, and the following primer pair ( 5′-CTAGAGCTCAGACACAGGAGATCCTGAGGATAC-3′ ; 5′-CTAGGATCCTCACATGGAGATTGAGTAAAGCTCTTC-3′ ).

Article Title: Partially Overlapping Primer-Based PCR for Genome Walking
Article Snippet: .. DNA manipulation and sequencing PCR products were purified with the Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China) or ligated into pMD18-T Vector or pMD19-T Simple Vector (TaKaRa). .. Then, the recombinant plasmids were transformed into E . coli DH5α cells according to TaKaRa’s guidelines, and were sequenced by Sangon Biotech Co., Ltd.

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    TaKaRa dna manipulation
    ACSL3 but not <t>ACSL6</t> is recruited into the Ct inclusion. HeLa cells were grown on coverslips and infected with Ct strain D at a MOI of 1 in cell culture medium without antibiotics and cycloheximide. Cells were fixed 36 hr post- infection. <t>DNA</t> was detected with Hoechst 33258 dye (blue). Bacteria were detected with a rabbit antibody against IncA, which was stained with an anti-rabbit antibody labeled with AlexaFluor®488 revealing the inclusion membrane (green, panels A to C) or with a mouse antibody against LPS, which was stained with Cy TM 3-conjugated anti-mouse antibody (red, panel D). Long-chain acyl-CoA synthetase 3, ACSL3, was detected with a mouse monoclonal antibody (red, panel B and C) and ACSL6 protein was detected with a rabbit antibody (green, panel D). The inclusion membrane of one representative inclusion is indicated with an arrow in panel A and B. Panel C displays a cropped portion of the image shown in panel B. Insets in panel C and D display the orthogonal (z, x) and (z, y) views of the deconvolved Z stack merged images. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in panels and insets represent 10 µm. Note that the two insets of panel C were slightly scaled up compare to the main image. Nuclei and inclusion are indicated with N and Inc, respectively. ACSL3 protein was detected inside the inclusion (panel B and C) and ACSL6 was not (panel D).
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    ACSL3 but not ACSL6 is recruited into the Ct inclusion. HeLa cells were grown on coverslips and infected with Ct strain D at a MOI of 1 in cell culture medium without antibiotics and cycloheximide. Cells were fixed 36 hr post- infection. DNA was detected with Hoechst 33258 dye (blue). Bacteria were detected with a rabbit antibody against IncA, which was stained with an anti-rabbit antibody labeled with AlexaFluor®488 revealing the inclusion membrane (green, panels A to C) or with a mouse antibody against LPS, which was stained with Cy TM 3-conjugated anti-mouse antibody (red, panel D). Long-chain acyl-CoA synthetase 3, ACSL3, was detected with a mouse monoclonal antibody (red, panel B and C) and ACSL6 protein was detected with a rabbit antibody (green, panel D). The inclusion membrane of one representative inclusion is indicated with an arrow in panel A and B. Panel C displays a cropped portion of the image shown in panel B. Insets in panel C and D display the orthogonal (z, x) and (z, y) views of the deconvolved Z stack merged images. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in panels and insets represent 10 µm. Note that the two insets of panel C were slightly scaled up compare to the main image. Nuclei and inclusion are indicated with N and Inc, respectively. ACSL3 protein was detected inside the inclusion (panel B and C) and ACSL6 was not (panel D).

    Journal: PLoS ONE

    Article Title: Eukaryotic Protein Recruitment into the Chlamydia Inclusion: Implications for Survival and Growth

    doi: 10.1371/journal.pone.0036843

    Figure Lengend Snippet: ACSL3 but not ACSL6 is recruited into the Ct inclusion. HeLa cells were grown on coverslips and infected with Ct strain D at a MOI of 1 in cell culture medium without antibiotics and cycloheximide. Cells were fixed 36 hr post- infection. DNA was detected with Hoechst 33258 dye (blue). Bacteria were detected with a rabbit antibody against IncA, which was stained with an anti-rabbit antibody labeled with AlexaFluor®488 revealing the inclusion membrane (green, panels A to C) or with a mouse antibody against LPS, which was stained with Cy TM 3-conjugated anti-mouse antibody (red, panel D). Long-chain acyl-CoA synthetase 3, ACSL3, was detected with a mouse monoclonal antibody (red, panel B and C) and ACSL6 protein was detected with a rabbit antibody (green, panel D). The inclusion membrane of one representative inclusion is indicated with an arrow in panel A and B. Panel C displays a cropped portion of the image shown in panel B. Insets in panel C and D display the orthogonal (z, x) and (z, y) views of the deconvolved Z stack merged images. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in panels and insets represent 10 µm. Note that the two insets of panel C were slightly scaled up compare to the main image. Nuclei and inclusion are indicated with N and Inc, respectively. ACSL3 protein was detected inside the inclusion (panel B and C) and ACSL6 was not (panel D).

    Article Snippet: DNA manipulation and transfection Full-length LPCAT1 cDNA and ACSL6 cDNA , were cloned into the pAcGFP1 vector (Clontech Laboratories, Mountain View, CA) to generate a fusion of the GFP protein to their amino-terminal extremity.

    Techniques: Infection, Cell Culture, Staining, Labeling, Microscopy

    LPCAT1 enzyme is tightly associated with the inclusion membrane. HeLa cells were grown on coverslips in cell culture medium without antibiotics and cycloheximide, and were transfected with a DNA construct expressing a GFP protein fused to acyl-CoA:lysophosphatidylcholine acyltransferase 1, LPCAT1. After 24 hr, cells were infected with Ct strain D at a MOI of 1 and were fixed 44 hr post-infection. DNA was detected with the Hoechst dye (blue) and the bacteria were detected with a rabbit antibody against IncA revealing the inclusion membrane (red). A deconvolved Z stack merged image with the GFP signal is shown in panel A. A cropped section of the image is displayed in the inset. White arrowheads indicate the position of the inclusion membrane. The tight association of LPCAT1with the inclusion membrane and overlap of the IncA and GFP signals, detected as yellow dots, can be observed in the inset. Panel B shows traces of the intensity of the signal for DNA, IncA and LPCAT1 (y axis) plotted in function of the distance (x axis) from area I to II as indicated by a red arrow on panel A. GFP and IncA signals co-localized in area I but not in area II. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in the panel and the inset represent 10 and 1 µm, respectively.

    Journal: PLoS ONE

    Article Title: Eukaryotic Protein Recruitment into the Chlamydia Inclusion: Implications for Survival and Growth

    doi: 10.1371/journal.pone.0036843

    Figure Lengend Snippet: LPCAT1 enzyme is tightly associated with the inclusion membrane. HeLa cells were grown on coverslips in cell culture medium without antibiotics and cycloheximide, and were transfected with a DNA construct expressing a GFP protein fused to acyl-CoA:lysophosphatidylcholine acyltransferase 1, LPCAT1. After 24 hr, cells were infected with Ct strain D at a MOI of 1 and were fixed 44 hr post-infection. DNA was detected with the Hoechst dye (blue) and the bacteria were detected with a rabbit antibody against IncA revealing the inclusion membrane (red). A deconvolved Z stack merged image with the GFP signal is shown in panel A. A cropped section of the image is displayed in the inset. White arrowheads indicate the position of the inclusion membrane. The tight association of LPCAT1with the inclusion membrane and overlap of the IncA and GFP signals, detected as yellow dots, can be observed in the inset. Panel B shows traces of the intensity of the signal for DNA, IncA and LPCAT1 (y axis) plotted in function of the distance (x axis) from area I to II as indicated by a red arrow on panel A. GFP and IncA signals co-localized in area I but not in area II. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in the panel and the inset represent 10 and 1 µm, respectively.

    Article Snippet: DNA manipulation and transfection Full-length LPCAT1 cDNA and ACSL6 cDNA , were cloned into the pAcGFP1 vector (Clontech Laboratories, Mountain View, CA) to generate a fusion of the GFP protein to their amino-terminal extremity.

    Techniques: Cell Culture, Transfection, Construct, Expressing, Infection, Microscopy

    Chromosome walking of the gadA locus of Lactobacillus brevis NCL912 (a), human aldolase A gene (b), malQ of Pichia pastoris GS115 (c), and hyg of rice (d). I: walking into 5’ regions of the genes (locus); II: walking into 3’ regions of the genes (locus). Each walking experiment contained four sets of PCRs that respectively utilized the four POP primer sets, POP1, POP2, POP3, and POP4, paired with a specific primer set. For each set of PCRs, only the results of secondary PCR (left lane) and tertiary (right lane) PCR are presented. White arrows indicate target bands. M1: DL2000 DNA marker. M2: λ-Hind III digest DNA Marker. M3: DL5000 DNA marker.

    Journal: PLoS ONE

    Article Title: Partially Overlapping Primer-Based PCR for Genome Walking

    doi: 10.1371/journal.pone.0120139

    Figure Lengend Snippet: Chromosome walking of the gadA locus of Lactobacillus brevis NCL912 (a), human aldolase A gene (b), malQ of Pichia pastoris GS115 (c), and hyg of rice (d). I: walking into 5’ regions of the genes (locus); II: walking into 3’ regions of the genes (locus). Each walking experiment contained four sets of PCRs that respectively utilized the four POP primer sets, POP1, POP2, POP3, and POP4, paired with a specific primer set. For each set of PCRs, only the results of secondary PCR (left lane) and tertiary (right lane) PCR are presented. White arrows indicate target bands. M1: DL2000 DNA marker. M2: λ-Hind III digest DNA Marker. M3: DL5000 DNA marker.

    Article Snippet: DNA manipulation and sequencing PCR products were purified with the Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China) or ligated into pMD18-T Vector or pMD19-T Simple Vector (TaKaRa).

    Techniques: Chromosome Walking, Polymerase Chain Reaction, Marker

    Overview of primer partially overlapping-based PCR. The first five high-stringency cycles (HSC) of each PCR are to increase copies of the single-stranded DNA of interest. The one low-stringency cycle (LSC) of primary PCR facilitates POP-P annealing to the target DNA and extension towards SP1. The one reduced-stringency cycle (RSC) of secondary PCR allowed POP-S to bind to the POP-P annealing site. A double-stranded target molecule was synthesized in the first HSC following LSC/RSC, and served as the template for the remaining HSCs; non-specific amplification was inhibited because the double-stranded form could not be obtained from a non-target single strand. Solid lines: the known sequence; dotted lines: the unknown sequence; thick black arrows with different heads: nested, specific primers; hollow arrows with different tails: POP primers; gray arrows: primers complementary sequences.

    Journal: PLoS ONE

    Article Title: Partially Overlapping Primer-Based PCR for Genome Walking

    doi: 10.1371/journal.pone.0120139

    Figure Lengend Snippet: Overview of primer partially overlapping-based PCR. The first five high-stringency cycles (HSC) of each PCR are to increase copies of the single-stranded DNA of interest. The one low-stringency cycle (LSC) of primary PCR facilitates POP-P annealing to the target DNA and extension towards SP1. The one reduced-stringency cycle (RSC) of secondary PCR allowed POP-S to bind to the POP-P annealing site. A double-stranded target molecule was synthesized in the first HSC following LSC/RSC, and served as the template for the remaining HSCs; non-specific amplification was inhibited because the double-stranded form could not be obtained from a non-target single strand. Solid lines: the known sequence; dotted lines: the unknown sequence; thick black arrows with different heads: nested, specific primers; hollow arrows with different tails: POP primers; gray arrows: primers complementary sequences.

    Article Snippet: DNA manipulation and sequencing PCR products were purified with the Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China) or ligated into pMD18-T Vector or pMD19-T Simple Vector (TaKaRa).

    Techniques: Polymerase Chain Reaction, Synthesized, Amplification, Sequencing

    Genome walking of the gadA locus of Lactobacillus brevis NCL912 and hyg of rice. Each walking experiment contained four sets of PCR reactions that respectively utilized the four SWPOP primer sets, SWPOP1 (I), SWPOP2 (II), SWPOP3 (III) and SWPOP4 (IV), paired with a specific primer set. For each set of PCR reactions, the results of primary PCR (P), secondary PCR (S) and tertiary PCR (T) are presented. White arrows indicate target bands. M: DL2000 DNA marker

    Journal: AMB Express

    Article Title: Stepwise partially overlapping primer-based PCR for genome walking

    doi: 10.1186/s13568-018-0610-7

    Figure Lengend Snippet: Genome walking of the gadA locus of Lactobacillus brevis NCL912 and hyg of rice. Each walking experiment contained four sets of PCR reactions that respectively utilized the four SWPOP primer sets, SWPOP1 (I), SWPOP2 (II), SWPOP3 (III) and SWPOP4 (IV), paired with a specific primer set. For each set of PCR reactions, the results of primary PCR (P), secondary PCR (S) and tertiary PCR (T) are presented. White arrows indicate target bands. M: DL2000 DNA marker

    Article Snippet: DNA manipulation and sequencing PCR products were purified with the MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, Dalian, China), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China).

    Techniques: Polymerase Chain Reaction, Marker

    Outline of stepwise partially overlapping primer-based PCR. The first five high-stringency cycles (HSCs) of each PCR were carried out to increase the copy number of specific single-stranded DNA (ssDNA) of interest with different length. The one low-stringency cycle (LSC) of primary PCR allowed SWPOP-P to anneal to the target DNA and extend towards SP-P. The one reduced-stringency cycle (RSC) of secondary/tertiary PCR allowed SWPOP-S/SWPOP-T to bind to the prior SWPOP complementary site(s). A double-stranded target DNA molecule was obtained in the first HSC following LSC/RSC, and served as the template for the remaining twenty-four HSCs. Non-target amplification was suppressed because the double-stranded form could not be synthesized from a non-target single strand. Drawings on the right side: potential nonspecific (non-target) amplifications; SP-P, SP-S, and SP-T: specific primer for primary, secondary, and tertiary PCR, respectively; SWPOP-P, SWPOP-S, and SWPOP-T: stepwise partially overlapping primer for primary, secondary, and tertiary PCR, respectively; solid lines: the known sequence; dotted lines: the unknown sequence; grey arrows: primers complementary sites

    Journal: AMB Express

    Article Title: Stepwise partially overlapping primer-based PCR for genome walking

    doi: 10.1186/s13568-018-0610-7

    Figure Lengend Snippet: Outline of stepwise partially overlapping primer-based PCR. The first five high-stringency cycles (HSCs) of each PCR were carried out to increase the copy number of specific single-stranded DNA (ssDNA) of interest with different length. The one low-stringency cycle (LSC) of primary PCR allowed SWPOP-P to anneal to the target DNA and extend towards SP-P. The one reduced-stringency cycle (RSC) of secondary/tertiary PCR allowed SWPOP-S/SWPOP-T to bind to the prior SWPOP complementary site(s). A double-stranded target DNA molecule was obtained in the first HSC following LSC/RSC, and served as the template for the remaining twenty-four HSCs. Non-target amplification was suppressed because the double-stranded form could not be synthesized from a non-target single strand. Drawings on the right side: potential nonspecific (non-target) amplifications; SP-P, SP-S, and SP-T: specific primer for primary, secondary, and tertiary PCR, respectively; SWPOP-P, SWPOP-S, and SWPOP-T: stepwise partially overlapping primer for primary, secondary, and tertiary PCR, respectively; solid lines: the known sequence; dotted lines: the unknown sequence; grey arrows: primers complementary sites

    Article Snippet: DNA manipulation and sequencing PCR products were purified with the MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa, Dalian, China), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China).

    Techniques: Polymerase Chain Reaction, Amplification, Synthesized, Sequencing