psti  (New England Biolabs)


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    PstI 50 000 units
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    Restriction Enzymes
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    New England Biolabs psti
    PstI
    PstI 50 000 units
    https://www.bioz.com/result/psti/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    psti - by Bioz Stars, 2020-02
    95/100 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 "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

    4) 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

    5) 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

    6) 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

    7) 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

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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:

    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

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    Acetylene Reduction Assay:

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    Amplification:

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    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
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    Article Title: One-step generation of conditional and reversible gene knockouts
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    Positive Control:

    Article Title: New STAT3-FOXL2 pathway and its function in cancer cells
    Article Snippet: The scrambled Silencer Select Negative Control #1 siRNA (Catalogue #4390843), Silencer Select GAPDH Positive Control (Catalogue #4390849) and BLOCK-IT Alexa Fluro Red Fluorescent Control (Catalogue #14750100), both purchased from Thermo Fisher Scientific (Waltham, USA). .. Pst I (Catalogue #R0140T) and Xma I (Catalogue #R0180S) were purchased from NEB (USA), The inhibitor, WP1066, was purchased from Merck Company (Merck KGaA, Darmstadt, Germany), and dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) as a stock solution.

    Clone Assay:

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    Article Snippet: .. The fused PCR fragment was digested with NdeI and PstI enzymes and cloned into plasmid pTYB1 (New England Biolabs). .. The resulting plasmid was called pDnaB4 and was verified by restriction analysis and DNA sequencing.

    Article Title: One-step generation of conditional and reversible gene knockouts
    Article Snippet: .. The FLIP cassette including selection marker gene was then transferred to the vector pUC118 (3318, Clontech) using the restriction enzymes SacI (R0156S, NEB) and PstI (R0140S, NEB) and Mighty cloning (6027, Takara). .. Addition of homologous arms to the FLIP cassette – FLIP targeting vector generation Homologous arms around an intron insertion site were amplified by high fidelity Phusion DNA polymerase (M0530S, NEB).

    Cytometry:

    Article Title: Induction of autophagy-dependent apoptosis in cancer cells through activation of ER stress: an uncovered anti-cancer mechanism by anti-alcoholism drug disulfiram
    Article Snippet: The following antibodies and the dilutions used for immunofluorescence and flow cytometry analysis were: Alexa Fluor® 488-conjugated rabbit anti-cleaved Caspase-3 (Asp175) (D3E9) mAb (#9603) (1:50), and Phycoerythrin (PE) conjugated-rabbit anti-LC3B (D11) XP® mAb (#8899) (1:50), and anti-LC3B (#2775) (1:200) were purchased from Cell Signaling Technology (Danvers, MA, USA), Mouse anti-human XBP1s mAb (MAB4257) (1:1000) was purchased from R & D Systems Inc (Minneapolis, MN, USA), and R-PE-conjugated AffiniPureF(ab’)2 Fragment Goat Anti-mouse IgG (115-116-071) (1:3000) and FITC- conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (111-095-003) (1:2000) were purchased from Jackson ImmunoResearch Inc (West Grove, PA, USA). .. The restriction enzyme PstI (#R0140S) was purchased from New England Biolabs (Ipswich, MA, USA).

    Blocking Assay:

    Article Title: New STAT3-FOXL2 pathway and its function in cancer cells
    Article Snippet: The scrambled Silencer Select Negative Control #1 siRNA (Catalogue #4390843), Silencer Select GAPDH Positive Control (Catalogue #4390849) and BLOCK-IT Alexa Fluro Red Fluorescent Control (Catalogue #14750100), both purchased from Thermo Fisher Scientific (Waltham, USA). .. Pst I (Catalogue #R0140T) and Xma I (Catalogue #R0180S) were purchased from NEB (USA), The inhibitor, WP1066, was purchased from Merck Company (Merck KGaA, Darmstadt, Germany), and dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) as a stock solution.

    Electrophoresis:

    Article Title: Oncogenic HPV Types Infection in Adolescents and University Women from North Portugal: From Self-Sampling to Cancer Prevention
    Article Snippet: The amplified fragment was analyzed by electrophoresis in 1.5% (w/v) agarose gels stained with ethidium bromide and visualized under UV light. .. Each restriction endonuclease reaction was performed in a 20 μ L final volume reaction, using 5 μ L of MY09/11 PCR product, 2 μ L of 10x recommended restriction buffer, and 10 units of each restriction endonucleases: PstI (New England BioLabs, R0140S), HaeIII (Fermentas Inc., #ER0151, Canada), DdeI (New England BioLabs, R0175L), and RsaI (New England BioLabs, R0167S).

    Incubation:

    Article Title: GC skew defines distinct RNA polymerase pause sites in CpG island promoters
    Article Snippet: Restriction enzymes used for the analysis of the MYC and SIAH2 loci were PstI (NEB no. R0140) and NsiI (NEB no. R0127); P2RY2 was XmaI (NEB no. R0180) and BsaWI (NEB no. R0567). .. The P2RY2 digest was modified slightly by sequential digestion first with BsaWI for 30 min at 60°C followed by the addition of XmaI and a second 400 units of BsaWI and incubation overnight at 37°C.

    Article Title: The NRP1 migraine risk variant shows evidence of association with menstrual migraine
    Article Snippet: This involved amplification of the targeted loci using a standard PCR protocol followed by digestion with the restriction enzymes Pst I (NEB #R0140S) for rs12845494, and Bsm AI (NEB #R0529L) for rs2506142. .. The PCR product for rs12845494 was added to a master mix of Pst I enzyme with NEBuffer 3.1 and incubated at 37 °C for 16 h. For rs2056142, the PCR product was added to a master mix of Bsm AI with SmartCutter® and incubated for an hour at 55 °C.

    Article Title: Serotypes and Virulence Profiles of Non-O157 Shiga Toxin-Producing Escherichia coli Isolates from Bovine Farms ▿
    Article Snippet: .. The resulting PCR product (890 bp) was incubated in the PstI restriction enzyme (R0140; New England BioLabs) according to the manufacturer's instructions. .. The absence of any band in the amplicon was indicative of stx 2dact , with 504-bp and 386-bp bands indicative of stx 2 and stx 2c , respectively ( ).

    Article Title: The NRP1 migraine risk variant shows evidence of association with menstrual migraine
    Article Snippet: This involved amplification of the targeted loci using a standard PCR protocol followed by digestion with the restriction enzymes Pst I (NEB #R0140S) for rs12845494, and Bsm AI (NEB #R0529L) for rs2506142. .. The PCR product for rs12845494 was added to a master mix of Pst I enzyme with NEBuffer 3.1 and incubated at 37 °C for 16 h. For rs2056142, the PCR product was added to a master mix of Bsm AI with SmartCutter® and incubated for an hour at 55 °C.

    Mass Spectrometry:

    Article Title: The NRP1 migraine risk variant shows evidence of association with menstrual migraine
    Article Snippet: Detection of primer extension products was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. .. This involved amplification of the targeted loci using a standard PCR protocol followed by digestion with the restriction enzymes Pst I (NEB #R0140S) for rs12845494, and Bsm AI (NEB #R0529L) for rs2506142.

    Modification:

    Article Title: siRNA Pool Targeting Different Sites of Human Hepatitis B Surface Antigen Efficiently Inhibits HBV Infection
    Article Snippet: Dulbecco's modified eagle's medium (DMEM), penicillin G (5000Unit/ml), Trypsin-EDTA, Trizol, DNase I, Lipofectamine LTX™, Lipofectamine 2000, PureLink™ Viral RNA/DNA Mini Kit, Monoclonal mouse anti-HBsAg and Alexa Fluor 488 goat anti-mouse IgG were purchased from Invitrogen Life Technologies (Carlsbad, CA). .. Restriction enzymes (PstI, EcoRI, ClaI, BsrGI, SgfI and BglII) were purchased from New England Biolabs (Ipswich, MA).

    Article Title: New STAT3-FOXL2 pathway and its function in cancer cells
    Article Snippet: Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from HyClone (GE Healthcare Life Sciences, Logan, UT, USA). .. Pst I (Catalogue #R0140T) and Xma I (Catalogue #R0180S) were purchased from NEB (USA), The inhibitor, WP1066, was purchased from Merck Company (Merck KGaA, Darmstadt, Germany), and dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) as a stock solution.

    Article Title: GC skew defines distinct RNA polymerase pause sites in CpG island promoters
    Article Snippet: Restriction enzymes used for the analysis of the MYC and SIAH2 loci were PstI (NEB no. R0140) and NsiI (NEB no. R0127); P2RY2 was XmaI (NEB no. R0180) and BsaWI (NEB no. R0567). .. The P2RY2 digest was modified slightly by sequential digestion first with BsaWI for 30 min at 60°C followed by the addition of XmaI and a second 400 units of BsaWI and incubation overnight at 37°C.

    Western Blot:

    Article Title: Induction of autophagy-dependent apoptosis in cancer cells through activation of ER stress: an uncovered anti-cancer mechanism by anti-alcoholism drug disulfiram
    Article Snippet: The following rabbit monoclonal antibodies (mAb) used for Western blot analyses were purchased from Cell Signaling Technology (Danvers, MA, USA), and used at the following indicated dilutions: anti-LC3A/B (#4108) (1:1000), anti-human cleaved PARP (#9541) (1:1000), anti-human and anti-mouse ß-actin (#4970) (1:2000), anti-human eIF2α (#5324) (1:1000) and anti-peIF2α (#3398) (1:1000), anti-human XBP1s (D2C1F) (#12782) (1:1000) and goat anti-rabbit IgG, HRP-linked secondary antibody (#7074) (1:2000). .. The restriction enzyme PstI (#R0140S) was purchased from New England Biolabs (Ipswich, MA, USA).

    Flow Cytometry:

    Article Title: Induction of autophagy-dependent apoptosis in cancer cells through activation of ER stress: an uncovered anti-cancer mechanism by anti-alcoholism drug disulfiram
    Article Snippet: The following antibodies and the dilutions used for immunofluorescence and flow cytometry analysis were: Alexa Fluor® 488-conjugated rabbit anti-cleaved Caspase-3 (Asp175) (D3E9) mAb (#9603) (1:50), and Phycoerythrin (PE) conjugated-rabbit anti-LC3B (D11) XP® mAb (#8899) (1:50), and anti-LC3B (#2775) (1:200) were purchased from Cell Signaling Technology (Danvers, MA, USA), Mouse anti-human XBP1s mAb (MAB4257) (1:1000) was purchased from R & D Systems Inc (Minneapolis, MN, USA), and R-PE-conjugated AffiniPureF(ab’)2 Fragment Goat Anti-mouse IgG (115-116-071) (1:3000) and FITC- conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (111-095-003) (1:2000) were purchased from Jackson ImmunoResearch Inc (West Grove, PA, USA). .. The restriction enzyme PstI (#R0140S) was purchased from New England Biolabs (Ipswich, MA, USA).

    Ligation:

    Article Title: GC skew defines distinct RNA polymerase pause sites in CpG island promoters
    Article Snippet: Ligation efficiency between the distal enhancer and various restriction fragments was interrogated by quantitative real-time PCR using Taqman probes. .. Restriction enzymes used for the analysis of the MYC and SIAH2 loci were PstI (NEB no. R0140) and NsiI (NEB no. R0127); P2RY2 was XmaI (NEB no. R0180) and BsaWI (NEB no. R0567).

    Article Title: Oligomerized Pool ENgineering (OPEN): An “Open-Source” Protocol for Making Customized Zinc Finger Arrays
    Article Snippet: .. > CAUTION Care should be taken to avoid contaminating laboratory equipment and benches with bacteriophage Restriction enzymes (all from New England Biolabs): BamH I (cat. no. R0136S), Bbs I (cat. no R0539L), Bsa I (cat. no. R0533L), EcoR I (cat. no. R0101L), Hind III (cat. no. R0104L), Pst I (cat. no. R0140S), Sap I (cat. no. R0569L), Xba I (cat. no. R0145L) 10× restriction enzyme buffers (New England Biolabs, included with enzymes) T4 DNA Ligase and associated standard T4 DNA Ligase reaction buffer (New England Biolabs, cat. no. M0202S) Quick Ligation Kit (New England Biolabs, cat. no. M2200S) T4 Polynucleotide Kinase (New England Biolabs, cat. no. M0201S) Cloned Pfu polymerase and associated 10× reaction buffer (Stratagene, cat. no. 600159-81) Expand High-Fidelity thermostable polymerase and associated 10× Expand buffer with MgCl2 (Roche, cat. no. 11732641001) < ! .. > CRITICAL We have found the use of Expand enzyme to be critical for successful amplification of the zinc finger pools AccuGel 29:1 acrylamide:bis-acrylamide solution (National Diagnostics, cat. no. EC-852) < !

    Synthesized:

    Article Title: siRNA Pool Targeting Different Sites of Human Hepatitis B Surface Antigen Efficiently Inhibits HBV Infection
    Article Snippet: Restriction enzymes (PstI, EcoRI, ClaI, BsrGI, SgfI and BglII) were purchased from New England Biolabs (Ipswich, MA). .. HBsAg ELISA kits were from Abazyme Company (Needham, MA). siRNAs and primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA).

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: .. pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol. .. PCR Thermocycler Tabletop centrifuge Tube rotator BioMag Separator (Advanced Magnetics Inc.) Nanodrop Spectrophotometer.

    DNA Sequencing:

    Article Title: Fluorescence polarization assay for inhibitors of the kinase domain of receptor interacting protein 1
    Article Snippet: The human RIP1 kinase domain (aa 8–327) was amplified by PCR using Phusion High-Fidelity Polymerase (New England Biolabs), digested with EcoRI and PstI restriction enzymes (New England Biolabs), and ligated into the same sites in the pAcGHLT-A Baculovirus Transfer Vector (BD Biosciences) creating the pAcGHLT-A-GST-hRIP1 8–327 plasmid. .. The human RIP1 kinase domain (aa 8–327) was amplified by PCR using Phusion High-Fidelity Polymerase (New England Biolabs), digested with EcoRI and PstI restriction enzymes (New England Biolabs), and ligated into the same sites in the pAcGHLT-A Baculovirus Transfer Vector (BD Biosciences) creating the pAcGHLT-A-GST-hRIP1 8–327 plasmid.

    Article Title: Biosynthesis of Staphylococcus aureus Autoinducing Peptides by Using the Synechocystis DnaB Mini-Intein ▿
    Article Snippet: The DnaB gene fragments were verified by DNA sequencing, PCR amplified from the pGEM5-T plasmid clones, and fused together with overlap extension PCR ( ). .. The fused PCR fragment was digested with NdeI and PstI enzymes and cloned into plasmid pTYB1 (New England Biolabs).

    Sequencing:

    Article Title: New STAT3-FOXL2 pathway and its function in cancer cells
    Article Snippet: Sequence-specific Silencer Select siRNAs targeting human STAT3 mRNA (Catalogue #4390824/s743, sense: 5′-GCCUCAAGAUUGACCUAGATT − 3′, antisense: 5′-UCUAGGUCAAUCUUGAGGCCT-3′) or FOXL2 mRNA (Catalogue #4392420/s2068, sense: 5′-CGAAGUUCCCGUUCUACGATT-3′, antisense: 5′-UCGUAGAACGGGAACUUCGCG-3′) were purchased from Thermo Fisher Scientific (Waltham, USA). .. Pst I (Catalogue #R0140T) and Xma I (Catalogue #R0180S) were purchased from NEB (USA), The inhibitor, WP1066, was purchased from Merck Company (Merck KGaA, Darmstadt, Germany), and dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) as a stock solution.

    Article Title: Fluorescence polarization assay for inhibitors of the kinase domain of receptor interacting protein 1
    Article Snippet: The human RIP1 kinase domain (aa 8–327) was amplified by PCR using Phusion High-Fidelity Polymerase (New England Biolabs), digested with EcoRI and PstI restriction enzymes (New England Biolabs), and ligated into the same sites in the pAcGHLT-A Baculovirus Transfer Vector (BD Biosciences) creating the pAcGHLT-A-GST-hRIP1 8–327 plasmid. .. The human RIP1 kinase domain (aa 8–327) was amplified by PCR using Phusion High-Fidelity Polymerase (New England Biolabs), digested with EcoRI and PstI restriction enzymes (New England Biolabs), and ligated into the same sites in the pAcGHLT-A Baculovirus Transfer Vector (BD Biosciences) creating the pAcGHLT-A-GST-hRIP1 8–327 plasmid.

    Article Title: Mitochondrial Involvement in Vertebrate Speciation? The Case of Mito-nuclear Genetic Divergence in Chameleons
    Article Snippet: Restriction Fragment Length Polymorphism Analysis Restriction fragment length polymorphism (RFLP) was conducted after sequencing of 20 (N = 20) specimens in all genes (except for ETFA where fewer sequences were available) to identify the alleles in the tested samples. .. RFLP was performed in the following genes to determine allele compositions (see supplementary table S11 , Supplementary Material online, for restriction mix and conditions): POLRMT (mutation at position 1218)—both Taq α I (New England BioLabs #R0149S) and Mluc I (New England Biolabs #R0538S); MRPL30—Pst I (New England BioLabs #R0140S); ETFA—Hind III (Thermo #ER0501); and LYRM4—Apek I (New England BioLabs #R0643S).

    Immunofluorescence:

    Article Title: Induction of autophagy-dependent apoptosis in cancer cells through activation of ER stress: an uncovered anti-cancer mechanism by anti-alcoholism drug disulfiram
    Article Snippet: The following antibodies and the dilutions used for immunofluorescence and flow cytometry analysis were: Alexa Fluor® 488-conjugated rabbit anti-cleaved Caspase-3 (Asp175) (D3E9) mAb (#9603) (1:50), and Phycoerythrin (PE) conjugated-rabbit anti-LC3B (D11) XP® mAb (#8899) (1:50), and anti-LC3B (#2775) (1:200) were purchased from Cell Signaling Technology (Danvers, MA, USA), Mouse anti-human XBP1s mAb (MAB4257) (1:1000) was purchased from R & D Systems Inc (Minneapolis, MN, USA), and R-PE-conjugated AffiniPureF(ab’)2 Fragment Goat Anti-mouse IgG (115-116-071) (1:3000) and FITC- conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (111-095-003) (1:2000) were purchased from Jackson ImmunoResearch Inc (West Grove, PA, USA). .. The restriction enzyme PstI (#R0140S) was purchased from New England Biolabs (Ipswich, MA, USA).

    Mutagenesis:

    Article Title: Mitochondrial Involvement in Vertebrate Speciation? The Case of Mito-nuclear Genetic Divergence in Chameleons
    Article Snippet: .. RFLP was performed in the following genes to determine allele compositions (see supplementary table S11 , Supplementary Material online, for restriction mix and conditions): POLRMT (mutation at position 1218)—both Taq α I (New England BioLabs #R0149S) and Mluc I (New England Biolabs #R0538S); MRPL30—Pst I (New England BioLabs #R0140S); ETFA—Hind III (Thermo #ER0501); and LYRM4—Apek I (New England BioLabs #R0643S). ..

    Isolation:

    Article Title: GC skew defines distinct RNA polymerase pause sites in CpG island promoters
    Article Snippet: DNA was isolated by proteinase K (500 mg) digestion overnight at 65°C, followed by phenol:chloroform extraction and ethanol precipitation. .. Restriction enzymes used for the analysis of the MYC and SIAH2 loci were PstI (NEB no. R0140) and NsiI (NEB no. R0127); P2RY2 was XmaI (NEB no. R0180) and BsaWI (NEB no. R0567).

    Subcloning:

    Article Title: Oligomerized Pool ENgineering (OPEN): An “Open-Source” Protocol for Making Customized Zinc Finger Arrays
    Article Snippet: Bacterial strain KJBAC1 (F- lacIq Δ hisB463 Δ( gpt-proAB-arg-lac ) XIII zaj∷Tn10 ; strain available through Addgene; ); this strain is required for propagation and subcloning of pBAC-lacZ-derived B2H reporter plasmids. .. > CAUTION Care should be taken to avoid contaminating laboratory equipment and benches with bacteriophage Restriction enzymes (all from New England Biolabs): BamH I (cat. no. R0136S), Bbs I (cat. no R0539L), Bsa I (cat. no. R0533L), EcoR I (cat. no. R0101L), Hind III (cat. no. R0104L), Pst I (cat. no. R0140S), Sap I (cat. no. R0569L), Xba I (cat. no. R0145L) 10× restriction enzyme buffers (New England Biolabs, included with enzymes) T4 DNA Ligase and associated standard T4 DNA Ligase reaction buffer (New England Biolabs, cat. no. M0202S) Quick Ligation Kit (New England Biolabs, cat. no. M2200S) T4 Polynucleotide Kinase (New England Biolabs, cat. no. M0201S) Cloned Pfu polymerase and associated 10× reaction buffer (Stratagene, cat. no. 600159-81) Expand High-Fidelity thermostable polymerase and associated 10× Expand buffer with MgCl2 (Roche, cat. no. 11732641001) < !

    Purification:

    Article Title: Oligomerized Pool ENgineering (OPEN): An “Open-Source” Protocol for Making Customized Zinc Finger Arrays
    Article Snippet: > CAUTION Care should be taken to avoid contaminating laboratory equipment and benches with bacteriophage Restriction enzymes (all from New England Biolabs): BamH I (cat. no. R0136S), Bbs I (cat. no R0539L), Bsa I (cat. no. R0533L), EcoR I (cat. no. R0101L), Hind III (cat. no. R0104L), Pst I (cat. no. R0140S), Sap I (cat. no. R0569L), Xba I (cat. no. R0145L) 10× restriction enzyme buffers (New England Biolabs, included with enzymes) T4 DNA Ligase and associated standard T4 DNA Ligase reaction buffer (New England Biolabs, cat. no. M0202S) Quick Ligation Kit (New England Biolabs, cat. no. M2200S) T4 Polynucleotide Kinase (New England Biolabs, cat. no. M0201S) Cloned Pfu polymerase and associated 10× reaction buffer (Stratagene, cat. no. 600159-81) Expand High-Fidelity thermostable polymerase and associated 10× Expand buffer with MgCl2 (Roche, cat. no. 11732641001) < ! .. 10% (w/v) ammonium persulfate in ddH2 O (Fisher, cat. no. 7727-54-0; store indefinitely at -20°C) TEMED (Fisher, cat. no. BP150-100; store indefinitely at 4°C) 100% ethanol (Pharmco, cat. no. 111ACS200) 70% (v/v) ethanol in ddH2 O QIAprep Spin Miniprep kit (Qiagen, cat. no. 27106) QIAquick PCR Purification kit (Qiagen, cat. no. 28106) MinElute PCR Purification kit (Qiagen, cat. no. 28006) LB medium (Difco, cat. No. 244620) LB agar medium (Difco, cat. No 244520) 2xYT medium (Difco, cat. No. 244020) SOB medium (Difco, cat. No. 244310) SOC medium (SOB medium with 0.4% (v/v) glucose) Bacto-Agar (Difco, cat. no. 214010) Bacto-Tryptone (Difco, cat. no. 211705) Sterile 10% (v/v) glyercol and sterile 50% (v/v) glycerol both in ddH2O (100% glycerol – Fisher, cat. no. BP229-1) 10× M9 salts (Difco, cat. no. 248510) 20% (w/v) Glucose in ddH2O (Mallinckrodt Baker, cat. No. 4912-06) < !

    Polymerase Chain Reaction:

    Article Title: Oncogenic HPV Types Infection in Adolescents and University Women from North Portugal: From Self-Sampling to Cancer Prevention
    Article Snippet: .. Each restriction endonuclease reaction was performed in a 20 μ L final volume reaction, using 5 μ L of MY09/11 PCR product, 2 μ L of 10x recommended restriction buffer, and 10 units of each restriction endonucleases: PstI (New England BioLabs, R0140S), HaeIII (Fermentas Inc., #ER0151, Canada), DdeI (New England BioLabs, R0175L), and RsaI (New England BioLabs, R0167S). .. The restricted fragments were separated by electrophoresis on 3% agarose gels with ethidium bromide staining and visualized under ultraviolet light.

    Article Title: Fluorescence polarization assay for inhibitors of the kinase domain of receptor interacting protein 1
    Article Snippet: .. The human RIP1 kinase domain (aa 8–327) was amplified by PCR using Phusion High-Fidelity Polymerase (New England Biolabs), digested with EcoRI and PstI restriction enzymes (New England Biolabs), and ligated into the same sites in the pAcGHLT-A Baculovirus Transfer Vector (BD Biosciences) creating the pAcGHLT-A-GST-hRIP1 8–327 plasmid. .. DNA sequencing (Core Facility, Tufts University) verified the correct sequence of the plasmid.

    Article Title: Oligomerized Pool ENgineering (OPEN): An “Open-Source” Protocol for Making Customized Zinc Finger Arrays
    Article Snippet: > CAUTION Care should be taken to avoid contaminating laboratory equipment and benches with bacteriophage Restriction enzymes (all from New England Biolabs): BamH I (cat. no. R0136S), Bbs I (cat. no R0539L), Bsa I (cat. no. R0533L), EcoR I (cat. no. R0101L), Hind III (cat. no. R0104L), Pst I (cat. no. R0140S), Sap I (cat. no. R0569L), Xba I (cat. no. R0145L) 10× restriction enzyme buffers (New England Biolabs, included with enzymes) T4 DNA Ligase and associated standard T4 DNA Ligase reaction buffer (New England Biolabs, cat. no. M0202S) Quick Ligation Kit (New England Biolabs, cat. no. M2200S) T4 Polynucleotide Kinase (New England Biolabs, cat. no. M0201S) Cloned Pfu polymerase and associated 10× reaction buffer (Stratagene, cat. no. 600159-81) Expand High-Fidelity thermostable polymerase and associated 10× Expand buffer with MgCl2 (Roche, cat. no. 11732641001) < ! .. 10% (w/v) ammonium persulfate in ddH2 O (Fisher, cat. no. 7727-54-0; store indefinitely at -20°C) TEMED (Fisher, cat. no. BP150-100; store indefinitely at 4°C) 100% ethanol (Pharmco, cat. no. 111ACS200) 70% (v/v) ethanol in ddH2 O QIAprep Spin Miniprep kit (Qiagen, cat. no. 27106) QIAquick PCR Purification kit (Qiagen, cat. no. 28106) MinElute PCR Purification kit (Qiagen, cat. no. 28006) LB medium (Difco, cat. No. 244620) LB agar medium (Difco, cat. No 244520) 2xYT medium (Difco, cat. No. 244020) SOB medium (Difco, cat. No. 244310) SOC medium (SOB medium with 0.4% (v/v) glucose) Bacto-Agar (Difco, cat. no. 214010) Bacto-Tryptone (Difco, cat. no. 211705) Sterile 10% (v/v) glyercol and sterile 50% (v/v) glycerol both in ddH2O (100% glycerol – Fisher, cat. no. BP229-1) 10× M9 salts (Difco, cat. no. 248510) 20% (w/v) Glucose in ddH2O (Mallinckrodt Baker, cat. No. 4912-06) < !

    Article Title: The NRP1 migraine risk variant shows evidence of association with menstrual migraine
    Article Snippet: .. This involved amplification of the targeted loci using a standard PCR protocol followed by digestion with the restriction enzymes Pst I (NEB #R0140S) for rs12845494, and Bsm AI (NEB #R0529L) for rs2506142. .. The PCR product for rs12845494 was added to a master mix of Pst I enzyme with NEBuffer 3.1 and incubated at 37 °C for 16 h. For rs2056142, the PCR product was added to a master mix of Bsm AI with SmartCutter® and incubated for an hour at 55 °C.

    Article Title: Biosynthesis of Staphylococcus aureus Autoinducing Peptides by Using the Synechocystis DnaB Mini-Intein ▿
    Article Snippet: .. The fused PCR fragment was digested with NdeI and PstI enzymes and cloned into plasmid pTYB1 (New England Biolabs). .. The resulting plasmid was called pDnaB4 and was verified by restriction analysis and DNA sequencing.

    Article Title: Serotypes and Virulence Profiles of Non-O157 Shiga Toxin-Producing Escherichia coli Isolates from Bovine Farms ▿
    Article Snippet: .. The resulting PCR product (890 bp) was incubated in the PstI restriction enzyme (R0140; New England BioLabs) according to the manufacturer's instructions. .. The absence of any band in the amplicon was indicative of stx 2dact , with 504-bp and 386-bp bands indicative of stx 2 and stx 2c , respectively ( ).

    Article Title: The NRP1 migraine risk variant shows evidence of association with menstrual migraine
    Article Snippet: .. This involved amplification of the targeted loci using a standard PCR protocol followed by digestion with the restriction enzymes Pst I (NEB #R0140S) for rs12845494, and Bsm AI (NEB #R0529L) for rs2506142. .. The PCR product for rs12845494 was added to a master mix of Pst I enzyme with NEBuffer 3.1 and incubated at 37 °C for 16 h. For rs2056142, the PCR product was added to a master mix of Bsm AI with SmartCutter® and incubated for an hour at 55 °C.

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: .. pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol. .. PCR Thermocycler Tabletop centrifuge Tube rotator BioMag Separator (Advanced Magnetics Inc.) Nanodrop Spectrophotometer.

    Article Title: One-step generation of conditional and reversible gene knockouts
    Article Snippet: Replacement of dsRed was done through restriction digest excision using EcoRI (R3101S, NEB) and Acc65l (RO599S, NEB) followed by insertion of PCR amplified selection marker genes using Infusion cloning (638909, Clontech). .. The FLIP cassette including selection marker gene was then transferred to the vector pUC118 (3318, Clontech) using the restriction enzymes SacI (R0156S, NEB) and PstI (R0140S, NEB) and Mighty cloning (6027, Takara).

    Construct:

    Article Title: Oligomerized Pool ENgineering (OPEN): An “Open-Source” Protocol for Making Customized Zinc Finger Arrays
    Article Snippet: Bacterial strain KJ1C (F- Δ hisB463 Δ (gpt-proAB-arg-lac)XIII zaj ∷ Tn10 ); available by request from the Joung lab); this strain is required to construct B2H selection strains. .. > CAUTION Care should be taken to avoid contaminating laboratory equipment and benches with bacteriophage Restriction enzymes (all from New England Biolabs): BamH I (cat. no. R0136S), Bbs I (cat. no R0539L), Bsa I (cat. no. R0533L), EcoR I (cat. no. R0101L), Hind III (cat. no. R0104L), Pst I (cat. no. R0140S), Sap I (cat. no. R0569L), Xba I (cat. no. R0145L) 10× restriction enzyme buffers (New England Biolabs, included with enzymes) T4 DNA Ligase and associated standard T4 DNA Ligase reaction buffer (New England Biolabs, cat. no. M0202S) Quick Ligation Kit (New England Biolabs, cat. no. M2200S) T4 Polynucleotide Kinase (New England Biolabs, cat. no. M0201S) Cloned Pfu polymerase and associated 10× reaction buffer (Stratagene, cat. no. 600159-81) Expand High-Fidelity thermostable polymerase and associated 10× Expand buffer with MgCl2 (Roche, cat. no. 11732641001) < !

    IA:

    Article Title: siRNA Pool Targeting Different Sites of Human Hepatitis B Surface Antigen Efficiently Inhibits HBV Infection
    Article Snippet: Restriction enzymes (PstI, EcoRI, ClaI, BsrGI, SgfI and BglII) were purchased from New England Biolabs (Ipswich, MA). .. HBsAg ELISA kits were from Abazyme Company (Needham, MA). siRNAs and primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA).

    Plasmid Preparation:

    Article Title: siRNA Pool Targeting Different Sites of Human Hepatitis B Surface Antigen Efficiently Inhibits HBV Infection
    Article Snippet: psiSTRIKE™ vector kit was purchased from Promega corporation (Madison, WI). .. Restriction enzymes (PstI, EcoRI, ClaI, BsrGI, SgfI and BglII) were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Fluorescence polarization assay for inhibitors of the kinase domain of receptor interacting protein 1
    Article Snippet: .. The human RIP1 kinase domain (aa 8–327) was amplified by PCR using Phusion High-Fidelity Polymerase (New England Biolabs), digested with EcoRI and PstI restriction enzymes (New England Biolabs), and ligated into the same sites in the pAcGHLT-A Baculovirus Transfer Vector (BD Biosciences) creating the pAcGHLT-A-GST-hRIP1 8–327 plasmid. .. DNA sequencing (Core Facility, Tufts University) verified the correct sequence of the plasmid.

    Article Title: Induction of autophagy-dependent apoptosis in cancer cells through activation of ER stress: an uncovered anti-cancer mechanism by anti-alcoholism drug disulfiram
    Article Snippet: The restriction enzyme PstI (#R0140S) was purchased from New England Biolabs (Ipswich, MA, USA). .. The plasmid for retroviral transduction, pQCXI Puro DsRed-LC3-GFP (DsRed-LC3-GFP), was a gift from David Sabatini (Addgene plasmid #31182, Cambridge, MA, USA) [ ].

    Article Title: Biosynthesis of Staphylococcus aureus Autoinducing Peptides by Using the Synechocystis DnaB Mini-Intein ▿
    Article Snippet: .. The fused PCR fragment was digested with NdeI and PstI enzymes and cloned into plasmid pTYB1 (New England Biolabs). .. The resulting plasmid was called pDnaB4 and was verified by restriction analysis and DNA sequencing.

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: .. pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol. .. PCR Thermocycler Tabletop centrifuge Tube rotator BioMag Separator (Advanced Magnetics Inc.) Nanodrop Spectrophotometer.

    Article Title: One-step generation of conditional and reversible gene knockouts
    Article Snippet: .. The FLIP cassette including selection marker gene was then transferred to the vector pUC118 (3318, Clontech) using the restriction enzymes SacI (R0156S, NEB) and PstI (R0140S, NEB) and Mighty cloning (6027, Takara). .. Addition of homologous arms to the FLIP cassette – FLIP targeting vector generation Homologous arms around an intron insertion site were amplified by high fidelity Phusion DNA polymerase (M0530S, NEB).

    Software:

    Article Title: The NRP1 migraine risk variant shows evidence of association with menstrual migraine
    Article Snippet: SpectroTYPER software was used to automatically import and analyze the genotyping data with genotypes called based on the calculated mass of the extension products. .. This involved amplification of the targeted loci using a standard PCR protocol followed by digestion with the restriction enzymes Pst I (NEB #R0140S) for rs12845494, and Bsm AI (NEB #R0529L) for rs2506142.

    Real-time Polymerase Chain Reaction:

    Article Title: GC skew defines distinct RNA polymerase pause sites in CpG island promoters
    Article Snippet: Ligation efficiency between the distal enhancer and various restriction fragments was interrogated by quantitative real-time PCR using Taqman probes. .. Restriction enzymes used for the analysis of the MYC and SIAH2 loci were PstI (NEB no. R0140) and NsiI (NEB no. R0127); P2RY2 was XmaI (NEB no. R0180) and BsaWI (NEB no. R0567).

    Negative Control:

    Article Title: New STAT3-FOXL2 pathway and its function in cancer cells
    Article Snippet: The scrambled Silencer Select Negative Control #1 siRNA (Catalogue #4390843), Silencer Select GAPDH Positive Control (Catalogue #4390849) and BLOCK-IT Alexa Fluro Red Fluorescent Control (Catalogue #14750100), both purchased from Thermo Fisher Scientific (Waltham, USA). .. Pst I (Catalogue #R0140T) and Xma I (Catalogue #R0180S) were purchased from NEB (USA), The inhibitor, WP1066, was purchased from Merck Company (Merck KGaA, Darmstadt, Germany), and dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) as a stock solution.

    Selection:

    Article Title: Oligomerized Pool ENgineering (OPEN): An “Open-Source” Protocol for Making Customized Zinc Finger Arrays
    Article Snippet: Bacterial strain KJ1C (F- Δ hisB463 Δ (gpt-proAB-arg-lac)XIII zaj ∷ Tn10 ); available by request from the Joung lab); this strain is required to construct B2H selection strains. .. > CAUTION Care should be taken to avoid contaminating laboratory equipment and benches with bacteriophage Restriction enzymes (all from New England Biolabs): BamH I (cat. no. R0136S), Bbs I (cat. no R0539L), Bsa I (cat. no. R0533L), EcoR I (cat. no. R0101L), Hind III (cat. no. R0104L), Pst I (cat. no. R0140S), Sap I (cat. no. R0569L), Xba I (cat. no. R0145L) 10× restriction enzyme buffers (New England Biolabs, included with enzymes) T4 DNA Ligase and associated standard T4 DNA Ligase reaction buffer (New England Biolabs, cat. no. M0202S) Quick Ligation Kit (New England Biolabs, cat. no. M2200S) T4 Polynucleotide Kinase (New England Biolabs, cat. no. M0201S) Cloned Pfu polymerase and associated 10× reaction buffer (Stratagene, cat. no. 600159-81) Expand High-Fidelity thermostable polymerase and associated 10× Expand buffer with MgCl2 (Roche, cat. no. 11732641001) < !

    Article Title: One-step generation of conditional and reversible gene knockouts
    Article Snippet: .. The FLIP cassette including selection marker gene was then transferred to the vector pUC118 (3318, Clontech) using the restriction enzymes SacI (R0156S, NEB) and PstI (R0140S, NEB) and Mighty cloning (6027, Takara). .. Addition of homologous arms to the FLIP cassette – FLIP targeting vector generation Homologous arms around an intron insertion site were amplified by high fidelity Phusion DNA polymerase (M0530S, NEB).

    Enzyme-linked Immunosorbent Assay:

    Article Title: siRNA Pool Targeting Different Sites of Human Hepatitis B Surface Antigen Efficiently Inhibits HBV Infection
    Article Snippet: Restriction enzymes (PstI, EcoRI, ClaI, BsrGI, SgfI and BglII) were purchased from New England Biolabs (Ipswich, MA). .. HBsAg ELISA kits were from Abazyme Company (Needham, MA). siRNAs and primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA).

    Ethanol Precipitation:

    Article Title: GC skew defines distinct RNA polymerase pause sites in CpG island promoters
    Article Snippet: DNA was isolated by proteinase K (500 mg) digestion overnight at 65°C, followed by phenol:chloroform extraction and ethanol precipitation. .. Restriction enzymes used for the analysis of the MYC and SIAH2 loci were PstI (NEB no. R0140) and NsiI (NEB no. R0127); P2RY2 was XmaI (NEB no. R0180) and BsaWI (NEB no. R0567).

    Marker:

    Article Title: One-step generation of conditional and reversible gene knockouts
    Article Snippet: .. The FLIP cassette including selection marker gene was then transferred to the vector pUC118 (3318, Clontech) using the restriction enzymes SacI (R0156S, NEB) and PstI (R0140S, NEB) and Mighty cloning (6027, Takara). .. Addition of homologous arms to the FLIP cassette – FLIP targeting vector generation Homologous arms around an intron insertion site were amplified by high fidelity Phusion DNA polymerase (M0530S, NEB).

    Staining:

    Article Title: Oncogenic HPV Types Infection in Adolescents and University Women from North Portugal: From Self-Sampling to Cancer Prevention
    Article Snippet: The amplified fragment was analyzed by electrophoresis in 1.5% (w/v) agarose gels stained with ethidium bromide and visualized under UV light. .. Each restriction endonuclease reaction was performed in a 20 μ L final volume reaction, using 5 μ L of MY09/11 PCR product, 2 μ L of 10x recommended restriction buffer, and 10 units of each restriction endonucleases: PstI (New England BioLabs, R0140S), HaeIII (Fermentas Inc., #ER0151, Canada), DdeI (New England BioLabs, R0175L), and RsaI (New England BioLabs, R0167S).

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: .. pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol. .. PCR Thermocycler Tabletop centrifuge Tube rotator BioMag Separator (Advanced Magnetics Inc.) Nanodrop Spectrophotometer.

    Variant Assay:

    Article Title: Serotypes and Virulence Profiles of Non-O157 Shiga Toxin-Producing Escherichia coli Isolates from Bovine Farms ▿
    Article Snippet: Bands at 137 bp and 151 bp ( ) indicated the presence of the variant stx 2c or stx 2dact . .. The resulting PCR product (890 bp) was incubated in the PstI restriction enzyme (R0140; New England BioLabs) according to the manufacturer's instructions.

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    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: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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.

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

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

    doi: 10.1073/pnas.0914845107

    Figure Lengend 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.

    Article Snippet: After 22 amplification cycles, the reactions were separated into four aliquots for digestion with NcoI, PstI (New England Biolabs), both, or neither.

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

    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0177751

    Figure Lengend 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.

    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.

    Techniques: Electron Microscopy, Plasmid Preparation