psti  (New England Biolabs)


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    PstI
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    PstI 50 000 units
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    Restriction Enzymes
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    Structured Review

    New England Biolabs psti
    PstI
    PstI 50 000 units
    https://www.bioz.com/result/psti/product/New England Biolabs
    Average 99 stars, based on 56 article reviews
    Price from $9.99 to $1999.99
    psti - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "High-Frequency Variation of Purine Biosynthesis Genes Is a Mechanism of Success in Campylobacter jejuni"

    Article Title: High-Frequency Variation of Purine Biosynthesis Genes Is a Mechanism of Success in Campylobacter jejuni

    Journal: mBio

    doi: 10.1128/mBio.00612-15

    SILAC-based affinity capture of Cj1132c with purF DNA bait and effect of cj1132c deletion on spontaneous mutation rate. (A) Screen for DNA-binding proteins captured with purF DNA bait. Differentially labeled (light, [ 13 C 6 ]arginine, or heavy, [ 13 C 6 14 N 4 ]arginine) C. jejuni total protein lysates were incubated with biotinylated purF DNA or prsA DNA, respectively. A 1:1 light/heavy mixture of DNA/protein complexes was captured on streptavidin beads. Proteins were eluted from beads by digestion with PstI restriction enzyme, and trypsinized proteins were analyzed by gel electrophoresis liquid chromatography-mass spectrometry. Each dot represents an identified peptide. SILAC analysis discriminates the specificity of an interaction; peptides with a low heavy/light ratio indicate higher-affinity capture. Data are representative of two independent experiments statistically assessed by the Benjamini-Hochberg false discovery rate (FDR), and FDR Q significance values are indicated: *, Q = 0.024; **, Q = 0.014. (B) Representative mass spectra for SILAC-labeled captured peptides. Top, equal mass/charge ratio of light and heavy forms of PorA peptide indicating nonspecific interaction; bottom, the light form of Cj1132c is present in a ratio > 16.8 times higher than the heavy form, indicating a high probability of interaction with the direct repeat and hypervariable region of purF . (C) Frequency of emergence of spontaneous ciprofloxacin resistance (spontaneous mutation rate) of Δ cj1132c and complemented Δ cj1132c / cj1132c mutants in purF- T91del genetic background. The numbers of spontaneous ciprofloxacin-resistant mutants relative to total CFU per OD 600 equivalent are presented. Mean results with SEM from six independent experiments each with two technical replicates are shown: ****, P ≤ 0.0001.
    Figure Legend Snippet: SILAC-based affinity capture of Cj1132c with purF DNA bait and effect of cj1132c deletion on spontaneous mutation rate. (A) Screen for DNA-binding proteins captured with purF DNA bait. Differentially labeled (light, [ 13 C 6 ]arginine, or heavy, [ 13 C 6 14 N 4 ]arginine) C. jejuni total protein lysates were incubated with biotinylated purF DNA or prsA DNA, respectively. A 1:1 light/heavy mixture of DNA/protein complexes was captured on streptavidin beads. Proteins were eluted from beads by digestion with PstI restriction enzyme, and trypsinized proteins were analyzed by gel electrophoresis liquid chromatography-mass spectrometry. Each dot represents an identified peptide. SILAC analysis discriminates the specificity of an interaction; peptides with a low heavy/light ratio indicate higher-affinity capture. Data are representative of two independent experiments statistically assessed by the Benjamini-Hochberg false discovery rate (FDR), and FDR Q significance values are indicated: *, Q = 0.024; **, Q = 0.014. (B) Representative mass spectra for SILAC-labeled captured peptides. Top, equal mass/charge ratio of light and heavy forms of PorA peptide indicating nonspecific interaction; bottom, the light form of Cj1132c is present in a ratio > 16.8 times higher than the heavy form, indicating a high probability of interaction with the direct repeat and hypervariable region of purF . (C) Frequency of emergence of spontaneous ciprofloxacin resistance (spontaneous mutation rate) of Δ cj1132c and complemented Δ cj1132c / cj1132c mutants in purF- T91del genetic background. The numbers of spontaneous ciprofloxacin-resistant mutants relative to total CFU per OD 600 equivalent are presented. Mean results with SEM from six independent experiments each with two technical replicates are shown: ****, P ≤ 0.0001.

    Techniques Used: Mutagenesis, DNA Binding Assay, Labeling, Incubation, Nucleic Acid Electrophoresis, Liquid Chromatography, Mass Spectrometry

    2) Product Images from "The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism"

    Article Title: The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0914845107

    Protein and transcript expression patterns of pyruvate kinase M1/M2 isoforms in cells and tissues. ( A ) Total adult-mouse organ/tissue homogenates were used for Western blotting with the indicated antibodies. rM1 and rM2: Flag-tagged purified recombinant human PK isoforms. ( B ) Total cell lysates of five human cancer-cell lines were used for Western blotting with the indicated antibodies. ( C ) Primers annealing to exon 8 and exon 11, respectively, were used to amplify mouse or human PK-M transcripts. The alternative exons that encode the distinctive segments of PK-M1 and PK-M2 are indicated in ( black ) and ( gray ), respectively. To distinguish between PK-M1 (exon 9 included) and PK-M2 (exon 10 included) isoforms, the PCR products were cleaved with NcoI, PstI, or both. There is an additional NcoI site (*) 11 bp away from the 3′ end of mouse exon 11. ( D ) Mouse organs were freshly dissected and perfused with saline. Total RNA was analyzed by radioactive RT-PCR followed by digestion with NcoI (N), PstI (P), or both enzymes (NP), plus an uncut control (U). Numbered bands are as follows: 1: Uncut M1 (502 bp); 2: uncut M2 (502 bp); 2*: M2 cleaved with NcoI in exon 11 (491 bp); 3: Pst1-cleaved M2 5’ fragment (286 bp); 4: NcoI-cleaved M1 5′ fragment (245 bp); 5: NcoI-cleaved M1 3’ fragment (240 bp); 6: PstI-cleaved M2 3’ fragment (216 bp); 7: PstI + NcoI-cleaved M2 3’ fragment (205 bp). The %M1 was quantified from band 1 (M1) and bands 3 and 6 (M2) in each P lane. ( E ) RT-PCR and restriction digest analysis of total RNA from the indicated human cell lines. The bands are numbered as for the mouse RT-PCR products, but the sizes are different because of the positions of the primers; the sizes are as follows: 1: 398 bp; 2: 398 bp; 3: 185 bp; 4: 144 bp; 5: 248 bp; 6: 213 bp. Note that the PK-M1 bands in the P and U lanes migrate slightly above the PK-M2 bands, which is also the case for the mouse PK-M1 transcripts.
    Figure Legend Snippet: Protein and transcript expression patterns of pyruvate kinase M1/M2 isoforms in cells and tissues. ( A ) Total adult-mouse organ/tissue homogenates were used for Western blotting with the indicated antibodies. rM1 and rM2: Flag-tagged purified recombinant human PK isoforms. ( B ) Total cell lysates of five human cancer-cell lines were used for Western blotting with the indicated antibodies. ( C ) Primers annealing to exon 8 and exon 11, respectively, were used to amplify mouse or human PK-M transcripts. The alternative exons that encode the distinctive segments of PK-M1 and PK-M2 are indicated in ( black ) and ( gray ), respectively. To distinguish between PK-M1 (exon 9 included) and PK-M2 (exon 10 included) isoforms, the PCR products were cleaved with NcoI, PstI, or both. There is an additional NcoI site (*) 11 bp away from the 3′ end of mouse exon 11. ( D ) Mouse organs were freshly dissected and perfused with saline. Total RNA was analyzed by radioactive RT-PCR followed by digestion with NcoI (N), PstI (P), or both enzymes (NP), plus an uncut control (U). Numbered bands are as follows: 1: Uncut M1 (502 bp); 2: uncut M2 (502 bp); 2*: M2 cleaved with NcoI in exon 11 (491 bp); 3: Pst1-cleaved M2 5’ fragment (286 bp); 4: NcoI-cleaved M1 5′ fragment (245 bp); 5: NcoI-cleaved M1 3’ fragment (240 bp); 6: PstI-cleaved M2 3’ fragment (216 bp); 7: PstI + NcoI-cleaved M2 3’ fragment (205 bp). The %M1 was quantified from band 1 (M1) and bands 3 and 6 (M2) in each P lane. ( E ) RT-PCR and restriction digest analysis of total RNA from the indicated human cell lines. The bands are numbered as for the mouse RT-PCR products, but the sizes are different because of the positions of the primers; the sizes are as follows: 1: 398 bp; 2: 398 bp; 3: 185 bp; 4: 144 bp; 5: 248 bp; 6: 213 bp. Note that the PK-M1 bands in the P and U lanes migrate slightly above the PK-M2 bands, which is also the case for the mouse PK-M1 transcripts.

    Techniques Used: Expressing, Western Blot, Purification, Recombinant, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    3) Product Images from "Isolation and Identification of Rickettsia massiliae from Rhipicephalus sanguineus Ticks Collected in Arizona"

    Article Title: Isolation and Identification of Rickettsia massiliae from Rhipicephalus sanguineus Ticks Collected in Arizona

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00122-06

    RFLP typing of spotted fever group rickettsiae in R. sanguineus ticks. The 70- to 602-nucleotide fragment of the rOmpA gene was amplified using seminested PCR, followed by restriction enzyme digestion with RsaI (A) and PstI (B). Restriction patterns of
    Figure Legend Snippet: RFLP typing of spotted fever group rickettsiae in R. sanguineus ticks. The 70- to 602-nucleotide fragment of the rOmpA gene was amplified using seminested PCR, followed by restriction enzyme digestion with RsaI (A) and PstI (B). Restriction patterns of

    Techniques Used: Amplification, Polymerase Chain Reaction

    4) Product Images from "In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium"

    Article Title: In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177751

    Circularization of pBR322 (cohesive ends) and pUC19 (blunt ends) plasmids mediated by DdrC visualized by electron microscopy. Panel a: Control pBR322 DNA linearized by PstI. Panels b-e: pBR322 circularization mediated by DdrC. Panel f. Control pUC19 DNA linearized by Ssp11. Panels g-j: pUC19 circularization mediated by DdrC. 1 μM DdrC was mixed with 2 nM molecules of linear pBR322 or pUC19 plasmid, containing cohesive or blunt ends, respectively. The shapes are similar at 0.5 μM, 1 μM or 2 μM of DdrC. Magnification = 85,000. Some loci of plasmid circularization are indicated by arrows.
    Figure Legend Snippet: Circularization of pBR322 (cohesive ends) and pUC19 (blunt ends) plasmids mediated by DdrC visualized by electron microscopy. Panel a: Control pBR322 DNA linearized by PstI. Panels b-e: pBR322 circularization mediated by DdrC. Panel f. Control pUC19 DNA linearized by Ssp11. Panels g-j: pUC19 circularization mediated by DdrC. 1 μM DdrC was mixed with 2 nM molecules of linear pBR322 or pUC19 plasmid, containing cohesive or blunt ends, respectively. The shapes are similar at 0.5 μM, 1 μM or 2 μM of DdrC. Magnification = 85,000. Some loci of plasmid circularization are indicated by arrows.

    Techniques Used: Electron Microscopy, Plasmid Preparation

    5) Product Images from "Genome-Wide Mapping of DNA Strand Breaks"

    Article Title: Genome-Wide Mapping of DNA Strand Breaks

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017353

    dDIP enrichment of yeast telomeric DNA evaluated by Southern blot. Extracted yeast DNA was first digested by XhoI or PstI, two enzymes cutting once in the conserved telomere proximal Y' repeat element giving a≈1.2 kb and ≈1.0 kb terminal restriction fragment respectively. A probe covering part of the telomeric Y' fragment including the terminal 0.35 kb TG1-3 repeats was used to reveal the capture of telomeric DNA. To evaluate the telomeres immunoprecipitation efficiency by the dDIP technique, 30%, 40% and 50% of the input DNA before immunoprecipitation was applied to the gel. N+, DNA from uninduced cells end-labeled with dATP, biotin-dATP and TdT. N-, DNA from uninduced cells end-labeled with dATP, biotin-dATP without TdT.
    Figure Legend Snippet: dDIP enrichment of yeast telomeric DNA evaluated by Southern blot. Extracted yeast DNA was first digested by XhoI or PstI, two enzymes cutting once in the conserved telomere proximal Y' repeat element giving a≈1.2 kb and ≈1.0 kb terminal restriction fragment respectively. A probe covering part of the telomeric Y' fragment including the terminal 0.35 kb TG1-3 repeats was used to reveal the capture of telomeric DNA. To evaluate the telomeres immunoprecipitation efficiency by the dDIP technique, 30%, 40% and 50% of the input DNA before immunoprecipitation was applied to the gel. N+, DNA from uninduced cells end-labeled with dATP, biotin-dATP and TdT. N-, DNA from uninduced cells end-labeled with dATP, biotin-dATP without TdT.

    Techniques Used: Southern Blot, Immunoprecipitation, Labeling

    6) Product Images from "Cellular assays for studying the Fe-S cluster containing base excision repair glycosylase MUTYH and homologs"

    Article Title: Cellular assays for studying the Fe-S cluster containing base excision repair glycosylase MUTYH and homologs

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2017.12.006

    Schematic representation of the plasmid based bacterial cell assay to assay MutY-mediated OG:A repair. The restriction enzyme sites for BamHI, PstI and Bmtl are indicated on the pACYC177 (green) plasmid, and the insert duplex carrying the OG:A mispair is shown in pink. The representative agarose gel on the left shows the expected bands formed after Bmtl digestion of the recovered plasmids. To analyze MutY variants, muty - Ec ).
    Figure Legend Snippet: Schematic representation of the plasmid based bacterial cell assay to assay MutY-mediated OG:A repair. The restriction enzyme sites for BamHI, PstI and Bmtl are indicated on the pACYC177 (green) plasmid, and the insert duplex carrying the OG:A mispair is shown in pink. The representative agarose gel on the left shows the expected bands formed after Bmtl digestion of the recovered plasmids. To analyze MutY variants, muty - Ec ).

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis

    7) Product Images from "Stoichiometric incorporation of base substitutions at specific sites in supercoiled DNA and supercoiled recombination intermediates"

    Article Title: Stoichiometric incorporation of base substitutions at specific sites in supercoiled DNA and supercoiled recombination intermediates

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq674

    Preparing supercoiled DNA labeled with fluorescent dyes. ( A ) Schematic summary of the method. Single-strand circular att P DNA from Phagemid DNA A(–) is used as the template for a PCR reaction that will give rise to the ‘non-template’ strand that will define (flank) the gap over a selected portion of att P. The left side of the gap is defined by a primer that has a 5′ adapter sequence (not complementary to the template) encoding a BamHI site followed by a HindIII site and then DNA sequence complementary to the template on the left side of the gap. The right side of the gap is defined by a primer that has a 5′ adapter sequence encoding a BamHI site followed by a PstI site and then DNA sequence complementary to the template on the right side of the gap. The linear products of PCR amplification are cut with BamHI, circularized by ligating the annealed overhangs, and used to generate the B(–) family of phagemids. Circular single-strand DNA made from the B(–) family of phagemids [SSC Phage DNA B(-)] is linearized by annealing it with a 45-base oligonucleotide complementary to the HindIII–PstI region and cleaving with those two enzymes. The resulting linear single-strand DNA, which corresponds to the ‘top-strand’ of att P, is annealed to the single-strand circular DNA made from phagemid A(+), which corresponds to the ‘bottom-strand’ of att P [SSC Phage DNA A(+)]. Included in the annealing mixture are oligonucleotides that will fill in the designed gap and will also bring in the specified acceptor (A) and/or donor (D) fluorescent dyes. The resulting nicked circle is incubated with ligase to generate covalently closed circular DNA that is then supercoiled by incubation with DNA gyrase to generate the final dye-labeled DNA. ( B ) Electrophoresis of the DNA intermediates and products in a 1% agarose gel with (lanes 1–5), or without (lanes 6–8), ethidium bromide (0.5 μg/ml). Following electrophoresis the DNA was visualized by staining with ethidium bromide. A 1-kb ladder (NEB) (lane 1) and a supercoiled plasmid DNA (lane 8) serve as markers. Lane 2, single-strand circular phage DNA [A(+) SSC], encoding the bottom strand of att P (from pMM12); lane 3, linearized phage DNA [B(–) linear], encoding truncated top-strand sequences of att P (from pMM32); lane 4, the nicked circle resulting from annealing the linearized B(–) DNA and the circular A(+) DNA, along with the gap-filling oligonucleotides; lane 5, the mixture of covalently closed and nicked circles after incubation with ligase; lane 6, the DNA from lane 5 electrophoresed in the absence of ethidium bromide; lane 7, the mixture of supercoiled and nicked DNA following incubation with DNA gyrase.
    Figure Legend Snippet: Preparing supercoiled DNA labeled with fluorescent dyes. ( A ) Schematic summary of the method. Single-strand circular att P DNA from Phagemid DNA A(–) is used as the template for a PCR reaction that will give rise to the ‘non-template’ strand that will define (flank) the gap over a selected portion of att P. The left side of the gap is defined by a primer that has a 5′ adapter sequence (not complementary to the template) encoding a BamHI site followed by a HindIII site and then DNA sequence complementary to the template on the left side of the gap. The right side of the gap is defined by a primer that has a 5′ adapter sequence encoding a BamHI site followed by a PstI site and then DNA sequence complementary to the template on the right side of the gap. The linear products of PCR amplification are cut with BamHI, circularized by ligating the annealed overhangs, and used to generate the B(–) family of phagemids. Circular single-strand DNA made from the B(–) family of phagemids [SSC Phage DNA B(-)] is linearized by annealing it with a 45-base oligonucleotide complementary to the HindIII–PstI region and cleaving with those two enzymes. The resulting linear single-strand DNA, which corresponds to the ‘top-strand’ of att P, is annealed to the single-strand circular DNA made from phagemid A(+), which corresponds to the ‘bottom-strand’ of att P [SSC Phage DNA A(+)]. Included in the annealing mixture are oligonucleotides that will fill in the designed gap and will also bring in the specified acceptor (A) and/or donor (D) fluorescent dyes. The resulting nicked circle is incubated with ligase to generate covalently closed circular DNA that is then supercoiled by incubation with DNA gyrase to generate the final dye-labeled DNA. ( B ) Electrophoresis of the DNA intermediates and products in a 1% agarose gel with (lanes 1–5), or without (lanes 6–8), ethidium bromide (0.5 μg/ml). Following electrophoresis the DNA was visualized by staining with ethidium bromide. A 1-kb ladder (NEB) (lane 1) and a supercoiled plasmid DNA (lane 8) serve as markers. Lane 2, single-strand circular phage DNA [A(+) SSC], encoding the bottom strand of att P (from pMM12); lane 3, linearized phage DNA [B(–) linear], encoding truncated top-strand sequences of att P (from pMM32); lane 4, the nicked circle resulting from annealing the linearized B(–) DNA and the circular A(+) DNA, along with the gap-filling oligonucleotides; lane 5, the mixture of covalently closed and nicked circles after incubation with ligase; lane 6, the DNA from lane 5 electrophoresed in the absence of ethidium bromide; lane 7, the mixture of supercoiled and nicked DNA following incubation with DNA gyrase.

    Techniques Used: Labeling, Polymerase Chain Reaction, Sequencing, Amplification, Incubation, Electrophoresis, Agarose Gel Electrophoresis, Staining, Plasmid Preparation

    8) Product Images from "Impact of next-generation sequencing error on analysis of barcoded plasmid libraries of known complexity and sequence"

    Article Title: Impact of next-generation sequencing error on analysis of barcoded plasmid libraries of known complexity and sequence

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku607

    Experimental design and analytical workflow for analysis of the Illumina-compatible barcode. ( A ) Structure and sequence of the Illumina-compatible barcode insert cloned into the NsiI site of the pEF1α.γc lentiviral construct. The insert contained a PstI site, 32 bp of the Illumina adaptor sequence, a 16-bp random sequence that functioned as the lentiviral barcode and an 18-bp known sequence. Numbers indicate the position of every fifth random nucleotide in the barcode. The SOLiD-compatible barcode followed a similar configuration, with the insert containing a PstI site, 23 bp of the P1-T adaptor, a 15-bp random sequence for the lentiviral barcode and the internal adaptor. For both barcode configurations, the barcode regions were amplified with 10 PCR cycles using primers that introduced the adaptor sequences required for the Illumina or SOLiD platforms. ( B ) Strategy for analyzing sequence data for the Illumina-compatible barcode. Raw sequence reads were filtered using the known sequence immediately following the barcode at positions 17–30 to eliminate indel errors. The lentiviral barcode was trimmed to positions 2–16 to avoid errors at position 1. The number of unique barcode sequences was counted with and without phred score filtering (Q30), and with and without allowing one mismatch. For the SOLiD-compatible barcode, raw sequence reads were filtered using 10 internal adaptor sequences and the number of unique barcode sequences were counted with and without allowing one mismatch.
    Figure Legend Snippet: Experimental design and analytical workflow for analysis of the Illumina-compatible barcode. ( A ) Structure and sequence of the Illumina-compatible barcode insert cloned into the NsiI site of the pEF1α.γc lentiviral construct. The insert contained a PstI site, 32 bp of the Illumina adaptor sequence, a 16-bp random sequence that functioned as the lentiviral barcode and an 18-bp known sequence. Numbers indicate the position of every fifth random nucleotide in the barcode. The SOLiD-compatible barcode followed a similar configuration, with the insert containing a PstI site, 23 bp of the P1-T adaptor, a 15-bp random sequence for the lentiviral barcode and the internal adaptor. For both barcode configurations, the barcode regions were amplified with 10 PCR cycles using primers that introduced the adaptor sequences required for the Illumina or SOLiD platforms. ( B ) Strategy for analyzing sequence data for the Illumina-compatible barcode. Raw sequence reads were filtered using the known sequence immediately following the barcode at positions 17–30 to eliminate indel errors. The lentiviral barcode was trimmed to positions 2–16 to avoid errors at position 1. The number of unique barcode sequences was counted with and without phred score filtering (Q30), and with and without allowing one mismatch. For the SOLiD-compatible barcode, raw sequence reads were filtered using 10 internal adaptor sequences and the number of unique barcode sequences were counted with and without allowing one mismatch.

    Techniques Used: Sequencing, Clone Assay, Construct, Amplification, Polymerase Chain Reaction

    9) Product Images from "Comparative analysis of linker histone H1, MeCP2, and HMGD1 on nucleosome stability and target site accessibility"

    Article Title: Comparative analysis of linker histone H1, MeCP2, and HMGD1 on nucleosome stability and target site accessibility

    Journal: Scientific Reports

    doi: 10.1038/srep33186

    CAP-mediated higher order chromatin structures are susceptible to chromatin remodeling by ISWI. ( a ) Scheme of the 3055 bp DNA construct containing a 17-mer array of 601 NPS separated by 30 bp linker DNA. The array DNA comprised 17 repeats of a NPS harboring the Widom-601 nucleosome positioning sequence (dashed line). Numbers indicate positions of restriction enzyme sites with respect to the nucleosome boundary 46 . ( b ) Remodeled and non-remodeled (ISWI or no ISWI) chromatin samples were digested with BamHI. ImageQuant imaging software was used to measure the intensity of the full and digested array DNA. These values were used to determine the percent uncut. ( c ) After ISWI remodeling, chromatin samples were digested with PstI and imaged on an agarose gel. Digestion amount quantified using band intensity, accounting for non-saturated arrays and were compared to No CAP chromatin. Error bars are the mean ± standard deviation.
    Figure Legend Snippet: CAP-mediated higher order chromatin structures are susceptible to chromatin remodeling by ISWI. ( a ) Scheme of the 3055 bp DNA construct containing a 17-mer array of 601 NPS separated by 30 bp linker DNA. The array DNA comprised 17 repeats of a NPS harboring the Widom-601 nucleosome positioning sequence (dashed line). Numbers indicate positions of restriction enzyme sites with respect to the nucleosome boundary 46 . ( b ) Remodeled and non-remodeled (ISWI or no ISWI) chromatin samples were digested with BamHI. ImageQuant imaging software was used to measure the intensity of the full and digested array DNA. These values were used to determine the percent uncut. ( c ) After ISWI remodeling, chromatin samples were digested with PstI and imaged on an agarose gel. Digestion amount quantified using band intensity, accounting for non-saturated arrays and were compared to No CAP chromatin. Error bars are the mean ± standard deviation.

    Techniques Used: Construct, Sequencing, Imaging, Software, Agarose Gel Electrophoresis, Standard Deviation

    10) Product Images from "An association study between CHEK2 gene mutations and susceptibility to breast cancer"

    Article Title: An association study between CHEK2 gene mutations and susceptibility to breast cancer

    Journal: Comparative Clinical Pathology

    doi: 10.1007/s00580-017-2455-x

    a A normal DNA, B and H PCR product uncut with Pst1, C and K heterozygous mutant-type cut with Scrf1: 194 bp and 174 bp fragmented and E negative control (water) M DNA marker. b Heterozygous mutant-type: 194 bp and 174 bp fragment by screening of PCR products using restriction enzymes ScrfI and PstI; A1 , heterozygous patient; B1 , C , and D homozygous normal
    Figure Legend Snippet: a A normal DNA, B and H PCR product uncut with Pst1, C and K heterozygous mutant-type cut with Scrf1: 194 bp and 174 bp fragmented and E negative control (water) M DNA marker. b Heterozygous mutant-type: 194 bp and 174 bp fragment by screening of PCR products using restriction enzymes ScrfI and PstI; A1 , heterozygous patient; B1 , C , and D homozygous normal

    Techniques Used: Polymerase Chain Reaction, Mutagenesis, Negative Control, Marker

    11) Product Images from "Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing"

    Article Title: Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing

    Journal: Cell

    doi: 10.1016/j.cell.2015.03.027

    mNET-seq Profiles for PKM Alternative Splicing after PTBP1 Depletion, Related to Figure 4 (A) PKM exons 8–11 are illustrated. Exon 9 (green) and exon 10 (orange) are mutually exclusive. PCR primers indicated as black triangles. RT-PCR products were digested with indicated exon-specific restriction enzyme (NcoI or PstI). (B) mNET-seq data around mutually exclusive exons 9 and 10 of PKM . mNET-seq/S5P signals at 3′ end of exon 9 and exon 10 are shown by green and orange arrows, respectively. Transcription direction, black arrow. (C) Western blot of PTBP1 and tubulin from siPTBP1-treated HeLa cells. (D) PKM RT-PCR products from PTBP1-depleted HeLa nuclear RNA were digested by NcoI. (E) mNET-seq/S5P data over mutually exclusive exons 9 and 10 of PKM from siLuc and siPTBP1-treated HeLa cells (top), followed by expanded view around 5′SS of introns 10 and 11. S5P-peaks at 3′ ends of exons, orange asterisks. Transcription direction, black arrows.
    Figure Legend Snippet: mNET-seq Profiles for PKM Alternative Splicing after PTBP1 Depletion, Related to Figure 4 (A) PKM exons 8–11 are illustrated. Exon 9 (green) and exon 10 (orange) are mutually exclusive. PCR primers indicated as black triangles. RT-PCR products were digested with indicated exon-specific restriction enzyme (NcoI or PstI). (B) mNET-seq data around mutually exclusive exons 9 and 10 of PKM . mNET-seq/S5P signals at 3′ end of exon 9 and exon 10 are shown by green and orange arrows, respectively. Transcription direction, black arrow. (C) Western blot of PTBP1 and tubulin from siPTBP1-treated HeLa cells. (D) PKM RT-PCR products from PTBP1-depleted HeLa nuclear RNA were digested by NcoI. (E) mNET-seq/S5P data over mutually exclusive exons 9 and 10 of PKM from siLuc and siPTBP1-treated HeLa cells (top), followed by expanded view around 5′SS of introns 10 and 11. S5P-peaks at 3′ ends of exons, orange asterisks. Transcription direction, black arrows.

    Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

    12) Product Images from "Transposable Prophage Mu Is Organized as a Stable Chromosomal Domain of E. coli"

    Article Title: Transposable Prophage Mu Is Organized as a Stable Chromosomal Domain of E. coli

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003902

    Interaction of prophage Mu ends probed by 3C methodology. ( A ) Experimental design (see text and Materials and Methods ). Blue line, Mu DNA; black line, E. coli DNA; purple and green dots, PstI and EcoRI sites, respectively; small arrows, primers used to amplify the DNA ligation product; red circle, paired L and R ends. ( B ) PCR products of ligation. Left: Primers were designed to produce a 155 bp fragment after PstI digestion-ligation (lane 2, arrowhead), and a 195 bp fragment after EcoRI digestion-ligation (lane 6, arrowhead); the fainter bands above the specific products in lanes 2 and 6 could not be re-amplified, hence are non-specific. The specific products were not observed in uncrosslinked (lanes 1, 5) and unligated (lanes 4, 8) samples. The band migrating at ∼100 bp in these lanes is non-specific. Lane 3, 7 DNA size marker ladder. ( C ) Quantitation of the ligation products. The qPCR signal obtained from wild-type MU ligation was set at 1. Crosslinking efficiency is defined as the ratio of qPCR signal from the ligation product in the ΔSGS strain compared to that in its wild-type parent. NL is the signal obtained from the non-ligated, crosslinked product in the wild-type reactions shown in lanes 4 and 8, and ΔMu is a similar control in a strain where Mu has been excised from ZL524 via recombination of the flanking loxP sites. The same set of primer pairs were used for all strains in either the PstI or the EcoRI panels. MU (ZL524), ΔSGS (ZL562), ΔMu (ZL580). ( D ) In vitro Cre- loxP recombination of the cross-linked DNA from the indicated strains before digestion with restriction enzymes.
    Figure Legend Snippet: Interaction of prophage Mu ends probed by 3C methodology. ( A ) Experimental design (see text and Materials and Methods ). Blue line, Mu DNA; black line, E. coli DNA; purple and green dots, PstI and EcoRI sites, respectively; small arrows, primers used to amplify the DNA ligation product; red circle, paired L and R ends. ( B ) PCR products of ligation. Left: Primers were designed to produce a 155 bp fragment after PstI digestion-ligation (lane 2, arrowhead), and a 195 bp fragment after EcoRI digestion-ligation (lane 6, arrowhead); the fainter bands above the specific products in lanes 2 and 6 could not be re-amplified, hence are non-specific. The specific products were not observed in uncrosslinked (lanes 1, 5) and unligated (lanes 4, 8) samples. The band migrating at ∼100 bp in these lanes is non-specific. Lane 3, 7 DNA size marker ladder. ( C ) Quantitation of the ligation products. The qPCR signal obtained from wild-type MU ligation was set at 1. Crosslinking efficiency is defined as the ratio of qPCR signal from the ligation product in the ΔSGS strain compared to that in its wild-type parent. NL is the signal obtained from the non-ligated, crosslinked product in the wild-type reactions shown in lanes 4 and 8, and ΔMu is a similar control in a strain where Mu has been excised from ZL524 via recombination of the flanking loxP sites. The same set of primer pairs were used for all strains in either the PstI or the EcoRI panels. MU (ZL524), ΔSGS (ZL562), ΔMu (ZL580). ( D ) In vitro Cre- loxP recombination of the cross-linked DNA from the indicated strains before digestion with restriction enzymes.

    Techniques Used: DNA Ligation, Polymerase Chain Reaction, Ligation, Amplification, Marker, Quantitation Assay, Real-time Polymerase Chain Reaction, In Vitro

    13) Product Images from "Comparison of emm Typing and Ribotyping with Three Restriction Enzymes To Characterize Clinical Isolates of Streptococcus pyogenes"

    Article Title: Comparison of emm Typing and Ribotyping with Three Restriction Enzymes To Characterize Clinical Isolates of Streptococcus pyogenes

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.43.1.150-155.2005

    Dendrogram of ribogroups P, C1, W, X, Z, I, C, T, O, J, G, A, N, E, B, V, and D are shown as representative groups. VCA shows the EcoRI pattern, and VCB and SEC-01 show PstI and HindIII patterns, respectively. The dendrogram is a composite of all three
    Figure Legend Snippet: Dendrogram of ribogroups P, C1, W, X, Z, I, C, T, O, J, G, A, N, E, B, V, and D are shown as representative groups. VCA shows the EcoRI pattern, and VCB and SEC-01 show PstI and HindIII patterns, respectively. The dendrogram is a composite of all three

    Techniques Used: Size-exclusion Chromatography

    Examples of riboprint patterns. Comparison of EcoRI, PstI, and HindIII riboprint patterns for common ribogroups is shown.
    Figure Legend Snippet: Examples of riboprint patterns. Comparison of EcoRI, PstI, and HindIII riboprint patterns for common ribogroups is shown.

    Techniques Used:

    14) Product Images from "Assessing the Amount of Quadruplex Structures Present within G2-Tract Synthetic Random-Sequence DNA Libraries"

    Article Title: Assessing the Amount of Quadruplex Structures Present within G2-Tract Synthetic Random-Sequence DNA Libraries

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064131

    Regeneration of G 2 N 5 population from PCR products. (A) Sequence of the PCR product. PstI and BamHI restriction sites are shown in boxes. Restriction enzyme cleavage sites are represented by arrows. (B) Digestion of amplified DGR36 sequence with BamHI and PstI individually (lanes 2 and 3) and in combination (lane 4). Coloured arrows indicate fragments comprised of sequences of the same colour in Panel A. Fragments consisting of only the first fifteen nucleotides of the 5′ regions of the PCR products in the single enzyme digestion experiments cannot be seen as only polymerized segments acquire 32 P during amplification.
    Figure Legend Snippet: Regeneration of G 2 N 5 population from PCR products. (A) Sequence of the PCR product. PstI and BamHI restriction sites are shown in boxes. Restriction enzyme cleavage sites are represented by arrows. (B) Digestion of amplified DGR36 sequence with BamHI and PstI individually (lanes 2 and 3) and in combination (lane 4). Coloured arrows indicate fragments comprised of sequences of the same colour in Panel A. Fragments consisting of only the first fifteen nucleotides of the 5′ regions of the PCR products in the single enzyme digestion experiments cannot be seen as only polymerized segments acquire 32 P during amplification.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Amplification

    15) Product Images from "Comparative analysis of linker histone H1, MeCP2, and HMGD1 on nucleosome stability and target site accessibility"

    Article Title: Comparative analysis of linker histone H1, MeCP2, and HMGD1 on nucleosome stability and target site accessibility

    Journal: Scientific Reports

    doi: 10.1038/srep33186

    CAP-mediated higher order chromatin structures are susceptible to chromatin remodeling by ISWI. ( a ) Scheme of the 3055 bp DNA construct containing a 17-mer array of 601 NPS separated by 30 bp linker DNA. The array DNA comprised 17 repeats of a NPS harboring the Widom-601 nucleosome positioning sequence (dashed line). Numbers indicate positions of restriction enzyme sites with respect to the nucleosome boundary 46 . ( b ) Remodeled and non-remodeled (ISWI or no ISWI) chromatin samples were digested with BamHI. ImageQuant imaging software was used to measure the intensity of the full and digested array DNA. These values were used to determine the percent uncut. ( c ) After ISWI remodeling, chromatin samples were digested with PstI and imaged on an agarose gel. Digestion amount quantified using band intensity, accounting for non-saturated arrays and were compared to No CAP chromatin. Error bars are the mean ± standard deviation.
    Figure Legend Snippet: CAP-mediated higher order chromatin structures are susceptible to chromatin remodeling by ISWI. ( a ) Scheme of the 3055 bp DNA construct containing a 17-mer array of 601 NPS separated by 30 bp linker DNA. The array DNA comprised 17 repeats of a NPS harboring the Widom-601 nucleosome positioning sequence (dashed line). Numbers indicate positions of restriction enzyme sites with respect to the nucleosome boundary 46 . ( b ) Remodeled and non-remodeled (ISWI or no ISWI) chromatin samples were digested with BamHI. ImageQuant imaging software was used to measure the intensity of the full and digested array DNA. These values were used to determine the percent uncut. ( c ) After ISWI remodeling, chromatin samples were digested with PstI and imaged on an agarose gel. Digestion amount quantified using band intensity, accounting for non-saturated arrays and were compared to No CAP chromatin. Error bars are the mean ± standard deviation.

    Techniques Used: Construct, Sequencing, Imaging, Software, Agarose Gel Electrophoresis, Standard Deviation

    16) Product Images from "Phenotypic and Molecular Analysis of Tellurite Resistance among Enterohemorrhagic Escherichia coli O157:H7 and Sorbitol-Fermenting O157:NM Clinical Isolates"

    Article Title: Phenotypic and Molecular Analysis of Tellurite Resistance among Enterohemorrhagic Escherichia coli O157:H7 and Sorbitol-Fermenting O157:NM Clinical Isolates

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.43.1.452-454.2005

    Hybridization of BamHI-PstI-digested genomic DNA from EHEC O157 strains and controls with the terC probe. M, molecular weight marker (1-kb DNA ladder; Gibco-BRL). In lanes 1 to 7, the following strains are displayed (serotype, Te-MIC in micrograms per
    Figure Legend Snippet: Hybridization of BamHI-PstI-digested genomic DNA from EHEC O157 strains and controls with the terC probe. M, molecular weight marker (1-kb DNA ladder; Gibco-BRL). In lanes 1 to 7, the following strains are displayed (serotype, Te-MIC in micrograms per

    Techniques Used: Hybridization, Molecular Weight, Marker

    17) Product Images from "Chimeric Genes in Deletions and Duplications Associated with Intellectual Disability"

    Article Title: Chimeric Genes in Deletions and Duplications Associated with Intellectual Disability

    Journal: International Journal of Genomics

    doi: 10.1155/2017/4798474

    (a) Sequences from both ends of the ZNF451-KIAA1586 chimera. (b) Digestion pattern of PCR amplicons of ZNF451, KIAA1586 and the chimera with RsaI and PstI. The size of each fragment is showed in brackets, the yellow box represents the segmental duplication, and PCR's primers are indicated as black arrows. (c) Schematic representation of chimeric gene according to these results.
    Figure Legend Snippet: (a) Sequences from both ends of the ZNF451-KIAA1586 chimera. (b) Digestion pattern of PCR amplicons of ZNF451, KIAA1586 and the chimera with RsaI and PstI. The size of each fragment is showed in brackets, the yellow box represents the segmental duplication, and PCR's primers are indicated as black arrows. (c) Schematic representation of chimeric gene according to these results.

    Techniques Used: Polymerase Chain Reaction

    Related Articles

    Amplification:

    Article Title: The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism
    Article Snippet: .. After 22 amplification cycles, the reactions were separated into four aliquots for digestion with NcoI, PstI (New England Biolabs), both, or neither. .. The products were analyzed on a 5% native polyacrylamide gel, visualized by autoradiography, and quantitated on a FUJIFILM FLA-5100 phosphoirmager (Fuji Medical Systems) using Multi Gauge software Version 2.3 (Fujifilm).

    Chromatography:

    Article Title: High-Frequency Variation of Purine Biosynthesis Genes Is a Mechanism of Success in Campylobacter jejuni
    Article Snippet: .. Protein-DNA complexes were pooled, eluted by PstI (NEB) restriction enzyme cleavage, and digested with ArgC, and purified peptides were analyzed by reverse-phase liquid chromatography-mass spectrometry. ..

    Isolation:

    Article Title: Cellular assays for studying the Fe-S cluster containing base excision repair glycosylase MUTYH and homologs
    Article Snippet: .. 10 μg of the isolated plasmid is digested, with 100 units each of BamHI and PstI (NEB) in the appropriate buffer (NEB 3.1) to generate sticky ends. .. The double digested plasmid (pACYC), purified using a 0.8% agarose gel (prepared in 1X TAE - 40m M Tris pH 7.6, 20 m M acetic acid and 1 m M EDTA pH 8.0) ( ) and extracted using Qiagen gel extraction kit, is ligated to 30 pmol of the insert duplex containing OG:A using 15 units of T4 ligase (Promega) followed by purification using a PCR clean-up kit (Qiagen).

    Labeling:

    Article Title: Relaxed Primer Specificity Associated with Reverse Transcriptases Encoded by the pFOXC Retroplasmids of Fusarium oxysporum
    Article Snippet: .. DNA standards were dephosphorylated PstI restriction fragments of bacteriophage λ that had been 5′ end labeled with [γ-32 P]ATP by using polynucleotide kinase (New England Biolabs, Beverly, Mass.). ..

    Purification:

    Article Title: Dynamics of the T4 Bacteriophage DNA Packasome Motor
    Article Snippet: .. The double dye AxC3Y-DNAs, single dye AxY-DNAs, and unlabeled Y-DNAs (90 bases each) were phosphorylated by T4 polynucleotide kinase (New England Biolabs), and the blunt ends were ligated to pBR322 DNA linearized with EcoRV, followed by PstI digestion and gel extraction to yield the Y-DNAs with the 3.7-kb purified pBR322 (New England Biolabs) derived leaders ( C ). .. Unmodified wild type empty large procapsids (elps) were prepared from the T4 procapsid producing mutant-infected bacteria.

    Article Title: High-Frequency Variation of Purine Biosynthesis Genes Is a Mechanism of Success in Campylobacter jejuni
    Article Snippet: .. Protein-DNA complexes were pooled, eluted by PstI (NEB) restriction enzyme cleavage, and digested with ArgC, and purified peptides were analyzed by reverse-phase liquid chromatography-mass spectrometry. ..

    Immunoprecipitation:

    Article Title: Genome-Wide Mapping of DNA Strand Breaks
    Article Snippet: .. Immunoprecipitation of yeast telomeres To match the detection levels observed by southern blots, we have immunoprecipitated 5 µg of uninduced yeast DNA previously digested with a ratio of 10 U per µg of DNA by XhoI and PstI (New England Biolabs). ..

    Plasmid Preparation:

    Article Title: In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium
    Article Snippet: .. To analyze interaction of DdrC with linear DNA fragment, pUC19 plasmid (New England Biolabs) was linearized with SspI (New England Biolabs) restriction enzyme producing blunt ends and pBR322 plasmid was linearized with PstI (New England Biolabs) producing 3’ overhang cohesive ends. .. For TEM experiments, all DNA molecules were purified on a MiniQ anion exchange column (GE Healthcare) with a chromatography SMART system.

    Article Title: Cellular assays for studying the Fe-S cluster containing base excision repair glycosylase MUTYH and homologs
    Article Snippet: .. 10 μg of the isolated plasmid is digested, with 100 units each of BamHI and PstI (NEB) in the appropriate buffer (NEB 3.1) to generate sticky ends. .. The double digested plasmid (pACYC), purified using a 0.8% agarose gel (prepared in 1X TAE - 40m M Tris pH 7.6, 20 m M acetic acid and 1 m M EDTA pH 8.0) ( ) and extracted using Qiagen gel extraction kit, is ligated to 30 pmol of the insert duplex containing OG:A using 15 units of T4 ligase (Promega) followed by purification using a PCR clean-up kit (Qiagen).

    Gel Extraction:

    Article Title: Dynamics of the T4 Bacteriophage DNA Packasome Motor
    Article Snippet: .. The double dye AxC3Y-DNAs, single dye AxY-DNAs, and unlabeled Y-DNAs (90 bases each) were phosphorylated by T4 polynucleotide kinase (New England Biolabs), and the blunt ends were ligated to pBR322 DNA linearized with EcoRV, followed by PstI digestion and gel extraction to yield the Y-DNAs with the 3.7-kb purified pBR322 (New England Biolabs) derived leaders ( C ). .. Unmodified wild type empty large procapsids (elps) were prepared from the T4 procapsid producing mutant-infected bacteria.

    Derivative Assay:

    Article Title: Dynamics of the T4 Bacteriophage DNA Packasome Motor
    Article Snippet: .. The double dye AxC3Y-DNAs, single dye AxY-DNAs, and unlabeled Y-DNAs (90 bases each) were phosphorylated by T4 polynucleotide kinase (New England Biolabs), and the blunt ends were ligated to pBR322 DNA linearized with EcoRV, followed by PstI digestion and gel extraction to yield the Y-DNAs with the 3.7-kb purified pBR322 (New England Biolabs) derived leaders ( C ). .. Unmodified wild type empty large procapsids (elps) were prepared from the T4 procapsid producing mutant-infected bacteria.

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    New England Biolabs hf psti high fidelity
    Adapter Design, PCR amplification of fragments. 1) The ligation product of a genomic DNA fragment (black) containing a <t>PstI</t> restriction site and a <t>MspI</t> restriction site. The forward adapter (blue) binds to a PstI generated overhang. The 4–9 bp barcode for this adapter is in bold with “X”. The MspI generated overhang corresponds to the reverse Y-adapter (green). The unpaired tail of the Y-adapter is underlined. 2) During the first round of PCR only the forward primer (red) can anneal. PCR synthesis of the complementary strand proceeds to the end of the fragment synthesizing the compliment of the Y-adapter tail. 3) During the second round of PCR the reverse primer (orange) can anneal to the newly synthesized compliment of the Y-adapter tail. This PCR reaction then proceeds to fill in the compliment of the forward adapter/primer on the other end of the same fragment.
    Hf Psti High Fidelity, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hf psti high fidelity/product/New England Biolabs
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    99
    New England Biolabs psti restriction fragments
    Pre- and posttreatment of endogenous reactions with DEAE-RT preparations. Reaction mixtures were preincubated in standard endogenous reaction buffer with or without actinomycin D (Ac) or RNase A (R) for 5 min prior to the addition of [ 32 P]dCTP. (Left panel) Reaction products were treated with RNase A (R) or DNase I (D) or left untreated prior to precipitation and electrophoresis in a 1.5% nondenaturing agarose gel. (Middle panel) Endogenous reaction mixtures were subjected to the following treatments: lane 6, mock incubation; lane 7, incubation in 0.1 M NaOH; lane 8, incubation with proteinase K; lane 9, incubation with proteinase K and then extraction with phenol-CIA; lane 10, mock incubation, followed by extraction with phenol-CIA. Reaction products were treated with glyoxal prior to electrophoresis. (Right panel) Endogenous reaction products were incubated with (lane 12) or without (lane 11) <t>λ</t> exonuclease. Reaction products were treated with glyoxal prior to electrophoresis. The filled arrows indicate the 1.95-kb cDNA reaction product, and the unfilled arrow indicates a band which is detected in nondenaturing gels and is more pronounced in reactions that retain endogenous RNA. The size of the products was determined by 5′-end-labeled <t>λ-PstI</t> and λ-HindIII/EcoRI restriction fragment markers (data not shown).
    Psti Restriction Fragments, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Adapter Design, PCR amplification of fragments. 1) The ligation product of a genomic DNA fragment (black) containing a PstI restriction site and a MspI restriction site. The forward adapter (blue) binds to a PstI generated overhang. The 4–9 bp barcode for this adapter is in bold with “X”. The MspI generated overhang corresponds to the reverse Y-adapter (green). The unpaired tail of the Y-adapter is underlined. 2) During the first round of PCR only the forward primer (red) can anneal. PCR synthesis of the complementary strand proceeds to the end of the fragment synthesizing the compliment of the Y-adapter tail. 3) During the second round of PCR the reverse primer (orange) can anneal to the newly synthesized compliment of the Y-adapter tail. This PCR reaction then proceeds to fill in the compliment of the forward adapter/primer on the other end of the same fragment.

    Journal: PLoS ONE

    Article Title: Development of High-Density Genetic Maps for Barley and Wheat Using a Novel Two-Enzyme Genotyping-by-Sequencing Approach

    doi: 10.1371/journal.pone.0032253

    Figure Lengend Snippet: Adapter Design, PCR amplification of fragments. 1) The ligation product of a genomic DNA fragment (black) containing a PstI restriction site and a MspI restriction site. The forward adapter (blue) binds to a PstI generated overhang. The 4–9 bp barcode for this adapter is in bold with “X”. The MspI generated overhang corresponds to the reverse Y-adapter (green). The unpaired tail of the Y-adapter is underlined. 2) During the first round of PCR only the forward primer (red) can anneal. PCR synthesis of the complementary strand proceeds to the end of the fragment synthesizing the compliment of the Y-adapter tail. 3) During the second round of PCR the reverse primer (orange) can anneal to the newly synthesized compliment of the Y-adapter tail. This PCR reaction then proceeds to fill in the compliment of the forward adapter/primer on the other end of the same fragment.

    Article Snippet: Restriction Digest: Genomic DNA (200 ng) was digested in 20 ul reaction volume of NEB Buffer 4 with 8 U of HF-PstI (High-Fidelity) and 8 U of MspI (New England BioLabs Inc., Ipswich, MA 01938).

    Techniques: Polymerase Chain Reaction, Amplification, Ligation, Generated, Synthesized

    Maps of doxycycline (dox)-inducible plasmids and over-expression analyses. A) Map of the pPID2 piggyBac cloning vector showing insulators in magenta; a DNA nuclear targeting sequence (DTS) in orange; multicloning sites (MCS) in purple; bacterial origin of replication (Ori) in cyan; bacterial β-lactamase (Bla) resistance gene (AmpR) in red; and piggyBac ITRs, IRES, and P2A sequences in grey. B) Map of the pPIDNB piggyBac dox-inducible vector showing restriction sites for cloning, coding sequences in yellow, terminator sequences in brown, and promoter sequences in green. pPIDNB constitutively expresses mNeonGreen (GFP) and coding sequences can be cloned into the plasmid under a bidirectional tetracycline (tet) inducible promoter using the AflII and PstI restriction sites. mScarlet-I, a red fluorescent protein (RFP), is expressed on the alternate side of the bidirectional tet promoter. C) Map of the pPIDNB2 vector, which is identical to pPIDNB except that GFP is localized to the nucleus using histone H2B. D) DF-1 cells transfected with pPIDNB constitutively express GFP and differentially express RFP in response to varying concentrations of dox after 24 hours. E) RFP-positive (i.e., dox-induced) cells relative to total number of GFP-positive (i.e., transfected) cells. F) dox induction was measured in DF-1 cells on the mRNA and G) protein levels for Cxcl14 (n = 2 for each group except n = 1 for the 2.5 ng/ml treatment; and mRNA level for H) Gas1 (n = 4 for each group). Levels are relative to 0 ng/ml of dox and normalized to 18S for mRNA and β-Actin for protein. I) Over-expression of Runx2 and J) Mmp13 with pPIDNB. DF-1 cells were transfected with control (cntrl) empty pPIDNB or pPIDNB plus Runx2 or Mmp13 coding sequence and treated with 50 ng/ml of dox for 24 hours. mRNA levels were normalized using 18S and protein using β-Actin. Representative WBs are shown below. N = 4 for each group. Error bars represent SEM. (*p

    Journal: bioRxiv

    Article Title: Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

    doi: 10.1101/2020.06.22.165704

    Figure Lengend Snippet: Maps of doxycycline (dox)-inducible plasmids and over-expression analyses. A) Map of the pPID2 piggyBac cloning vector showing insulators in magenta; a DNA nuclear targeting sequence (DTS) in orange; multicloning sites (MCS) in purple; bacterial origin of replication (Ori) in cyan; bacterial β-lactamase (Bla) resistance gene (AmpR) in red; and piggyBac ITRs, IRES, and P2A sequences in grey. B) Map of the pPIDNB piggyBac dox-inducible vector showing restriction sites for cloning, coding sequences in yellow, terminator sequences in brown, and promoter sequences in green. pPIDNB constitutively expresses mNeonGreen (GFP) and coding sequences can be cloned into the plasmid under a bidirectional tetracycline (tet) inducible promoter using the AflII and PstI restriction sites. mScarlet-I, a red fluorescent protein (RFP), is expressed on the alternate side of the bidirectional tet promoter. C) Map of the pPIDNB2 vector, which is identical to pPIDNB except that GFP is localized to the nucleus using histone H2B. D) DF-1 cells transfected with pPIDNB constitutively express GFP and differentially express RFP in response to varying concentrations of dox after 24 hours. E) RFP-positive (i.e., dox-induced) cells relative to total number of GFP-positive (i.e., transfected) cells. F) dox induction was measured in DF-1 cells on the mRNA and G) protein levels for Cxcl14 (n = 2 for each group except n = 1 for the 2.5 ng/ml treatment; and mRNA level for H) Gas1 (n = 4 for each group). Levels are relative to 0 ng/ml of dox and normalized to 18S for mRNA and β-Actin for protein. I) Over-expression of Runx2 and J) Mmp13 with pPIDNB. DF-1 cells were transfected with control (cntrl) empty pPIDNB or pPIDNB plus Runx2 or Mmp13 coding sequence and treated with 50 ng/ml of dox for 24 hours. mRNA levels were normalized using 18S and protein using β-Actin. Representative WBs are shown below. N = 4 for each group. Error bars represent SEM. (*p

    Article Snippet: Following confirmation of cloning of full length coding sequences by Sanger sequencing, Runx2 , Mmp13 , Cxcl14 , and Gas1 were cloned into pEPIC1.1 digested with AflII (NEB, R0520S) and EcoRI (NEB, R3101S) or pPIDNB digested with AflII (NEB, R0520S) and PstI (NEB, R3140S) using NEBuilder HiFi DNA Assembly Master Mix.

    Techniques: Over Expression, Clone Assay, Plasmid Preparation, Sequencing, Transfection

    Pre- and posttreatment of endogenous reactions with DEAE-RT preparations. Reaction mixtures were preincubated in standard endogenous reaction buffer with or without actinomycin D (Ac) or RNase A (R) for 5 min prior to the addition of [ 32 P]dCTP. (Left panel) Reaction products were treated with RNase A (R) or DNase I (D) or left untreated prior to precipitation and electrophoresis in a 1.5% nondenaturing agarose gel. (Middle panel) Endogenous reaction mixtures were subjected to the following treatments: lane 6, mock incubation; lane 7, incubation in 0.1 M NaOH; lane 8, incubation with proteinase K; lane 9, incubation with proteinase K and then extraction with phenol-CIA; lane 10, mock incubation, followed by extraction with phenol-CIA. Reaction products were treated with glyoxal prior to electrophoresis. (Right panel) Endogenous reaction products were incubated with (lane 12) or without (lane 11) λ exonuclease. Reaction products were treated with glyoxal prior to electrophoresis. The filled arrows indicate the 1.95-kb cDNA reaction product, and the unfilled arrow indicates a band which is detected in nondenaturing gels and is more pronounced in reactions that retain endogenous RNA. The size of the products was determined by 5′-end-labeled λ-PstI and λ-HindIII/EcoRI restriction fragment markers (data not shown).

    Journal: Eukaryotic Cell

    Article Title: Relaxed Primer Specificity Associated with Reverse Transcriptases Encoded by the pFOXC Retroplasmids of Fusarium oxysporum

    doi: 10.1128/EC.3.6.1589-1600.2004

    Figure Lengend Snippet: Pre- and posttreatment of endogenous reactions with DEAE-RT preparations. Reaction mixtures were preincubated in standard endogenous reaction buffer with or without actinomycin D (Ac) or RNase A (R) for 5 min prior to the addition of [ 32 P]dCTP. (Left panel) Reaction products were treated with RNase A (R) or DNase I (D) or left untreated prior to precipitation and electrophoresis in a 1.5% nondenaturing agarose gel. (Middle panel) Endogenous reaction mixtures were subjected to the following treatments: lane 6, mock incubation; lane 7, incubation in 0.1 M NaOH; lane 8, incubation with proteinase K; lane 9, incubation with proteinase K and then extraction with phenol-CIA; lane 10, mock incubation, followed by extraction with phenol-CIA. Reaction products were treated with glyoxal prior to electrophoresis. (Right panel) Endogenous reaction products were incubated with (lane 12) or without (lane 11) λ exonuclease. Reaction products were treated with glyoxal prior to electrophoresis. The filled arrows indicate the 1.95-kb cDNA reaction product, and the unfilled arrow indicates a band which is detected in nondenaturing gels and is more pronounced in reactions that retain endogenous RNA. The size of the products was determined by 5′-end-labeled λ-PstI and λ-HindIII/EcoRI restriction fragment markers (data not shown).

    Article Snippet: DNA standards were dephosphorylated PstI restriction fragments of bacteriophage λ that had been 5′ end labeled with [γ-32 P]ATP by using polynucleotide kinase (New England Biolabs, Beverly, Mass.).

    Techniques: Electrophoresis, Agarose Gel Electrophoresis, Incubation, Labeling

    SILAC-based affinity capture of Cj1132c with purF DNA bait and effect of cj1132c deletion on spontaneous mutation rate. (A) Screen for DNA-binding proteins captured with purF DNA bait. Differentially labeled (light, [ 13 C 6 ]arginine, or heavy, [ 13 C 6 14 N 4 ]arginine) C. jejuni total protein lysates were incubated with biotinylated purF DNA or prsA DNA, respectively. A 1:1 light/heavy mixture of DNA/protein complexes was captured on streptavidin beads. Proteins were eluted from beads by digestion with PstI restriction enzyme, and trypsinized proteins were analyzed by gel electrophoresis liquid chromatography-mass spectrometry. Each dot represents an identified peptide. SILAC analysis discriminates the specificity of an interaction; peptides with a low heavy/light ratio indicate higher-affinity capture. Data are representative of two independent experiments statistically assessed by the Benjamini-Hochberg false discovery rate (FDR), and FDR Q significance values are indicated: *, Q = 0.024; **, Q = 0.014. (B) Representative mass spectra for SILAC-labeled captured peptides. Top, equal mass/charge ratio of light and heavy forms of PorA peptide indicating nonspecific interaction; bottom, the light form of Cj1132c is present in a ratio > 16.8 times higher than the heavy form, indicating a high probability of interaction with the direct repeat and hypervariable region of purF . (C) Frequency of emergence of spontaneous ciprofloxacin resistance (spontaneous mutation rate) of Δ cj1132c and complemented Δ cj1132c / cj1132c mutants in purF- T91del genetic background. The numbers of spontaneous ciprofloxacin-resistant mutants relative to total CFU per OD 600 equivalent are presented. Mean results with SEM from six independent experiments each with two technical replicates are shown: ****, P ≤ 0.0001.

    Journal: mBio

    Article Title: High-Frequency Variation of Purine Biosynthesis Genes Is a Mechanism of Success in Campylobacter jejuni

    doi: 10.1128/mBio.00612-15

    Figure Lengend Snippet: SILAC-based affinity capture of Cj1132c with purF DNA bait and effect of cj1132c deletion on spontaneous mutation rate. (A) Screen for DNA-binding proteins captured with purF DNA bait. Differentially labeled (light, [ 13 C 6 ]arginine, or heavy, [ 13 C 6 14 N 4 ]arginine) C. jejuni total protein lysates were incubated with biotinylated purF DNA or prsA DNA, respectively. A 1:1 light/heavy mixture of DNA/protein complexes was captured on streptavidin beads. Proteins were eluted from beads by digestion with PstI restriction enzyme, and trypsinized proteins were analyzed by gel electrophoresis liquid chromatography-mass spectrometry. Each dot represents an identified peptide. SILAC analysis discriminates the specificity of an interaction; peptides with a low heavy/light ratio indicate higher-affinity capture. Data are representative of two independent experiments statistically assessed by the Benjamini-Hochberg false discovery rate (FDR), and FDR Q significance values are indicated: *, Q = 0.024; **, Q = 0.014. (B) Representative mass spectra for SILAC-labeled captured peptides. Top, equal mass/charge ratio of light and heavy forms of PorA peptide indicating nonspecific interaction; bottom, the light form of Cj1132c is present in a ratio > 16.8 times higher than the heavy form, indicating a high probability of interaction with the direct repeat and hypervariable region of purF . (C) Frequency of emergence of spontaneous ciprofloxacin resistance (spontaneous mutation rate) of Δ cj1132c and complemented Δ cj1132c / cj1132c mutants in purF- T91del genetic background. The numbers of spontaneous ciprofloxacin-resistant mutants relative to total CFU per OD 600 equivalent are presented. Mean results with SEM from six independent experiments each with two technical replicates are shown: ****, P ≤ 0.0001.

    Article Snippet: Protein-DNA complexes were pooled, eluted by PstI (NEB) restriction enzyme cleavage, and digested with ArgC, and purified peptides were analyzed by reverse-phase liquid chromatography-mass spectrometry.

    Techniques: Mutagenesis, DNA Binding Assay, Labeling, Incubation, Nucleic Acid Electrophoresis, Liquid Chromatography, Mass Spectrometry