pcr polymerase chain reaction clean up Search Results


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    Zymo Research zymoclean gel dna recovery kit
    Zymoclean Gel Dna Recovery Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 5708 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher genejet pcr purification kit
    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and <t>PCR</t> Clean-Up System (Promega); Th, <t>GeneJET</t> PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
    Genejet Pcr Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5354 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr clean up system
    Screening and generation of Mp erf13 mutants (A) The flow chart of the screen for mutants defective in oil body formation from <t>T-DNA-insertion</t> lines. (B) A maximum likelihood phylogenetic tree of proteins with one ERF/AP2 domain. The color code is shown in Fig S1A . The branch lengths are proportional to the estimated number of substitutions per site. Bootstrap probability is indicated as a percentage on each branch with at least 50% support. A more detailed tree was presented previously ( 20 ). (C) Schematic representation of the Mp ERF13 gene structure and mutations generated in this study. Gray and black boxes indicate the UTR and coding sequences, respectively. Asterisks with numbers indicate sites of designed gRNA to generate Mp erf13-1 ge (*1 and *4) and Mp erf13-2 ge (*2 and *3). (D) <t>PCR-based</t> genotyping of Tak-1, Mp erf13 GOF , Mp erf13-1 ge , and Mp erf13-2 ge . The combinations and annealing sites of primers (a to d) are shown in (C). (E) The genomic and predicted amino acid sequences of the Mp ERF13 locus in Tak-1 and Mp erf13 mutants. Light blue, dark blue, and magenta letters indicate UTR, coding, and predicted amino acid sequences, respectively. The PAM sequences for gRNAs are underlined. The caret indicates the indel site. (F) Fluorescent and bright-field images of BODIPY-stained three-week-old thalli of Tak-1, Mp erf13-1 ge , and Mp erf13-2 ge . Bars = 0.5 mm. (G) The number of oil bodies visualized with BODIPY in a unit area (2.0 mm × 2.0 mm). Bars indicate means ± SD. Statistical analyses between Tak-1 and each genotype were conducted using a two-tailed Welch’s t -test. Sample numbers were 23 thalli for Tak-1, 24 for Mp erf13-1 ge , and 26 for Mp erf13-2 ge . p -values are 5.14×10 −11 for Mp erf13-1 ge and 5.14×10 −11 for Mp erf13-2 ge .
    Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 22798 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL pcr clean up kit
    Host and viral <t>DNA</t> content of differently treated ASFV samples. Quantitative dual <t>PCR</t> was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.
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    Thermo Fisher purelink pcr purification kit
    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and <t>PCR</t> Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, <t>PureLink®</t> PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
    Purelink Pcr Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4057 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega wizard sv gel
    Illustrative representation of HSP 70 (234) amplification for Leishmania sp. before and after re-amplification of the <t>PCR</t> products. (A) PCR products of first amplification of HSP 70 (234) targeting analyzed by electrophoresis polyacrylamide gel stained with silver. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Infected Thrichomys laurentius from São Raimundo Nonato/PI; 2. Negative control of PCR reaction. (B) PCR products of re-amplification of the product obtained in the first PCR reaction. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Negative Thrichomys fosteri from Corumbá/MS (Positive only in kDNA); 2–5. Infected T. laurentius from São Raimundo Nonato/PI; 6. Infected T. fosteri from Corumbá/MS; 7. Negative control of PCR after re-amplification.
    Wizard Sv Gel, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 4009 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore genelute pcr clean up kit
    Illustrative representation of HSP 70 (234) amplification for Leishmania sp. before and after re-amplification of the <t>PCR</t> products. (A) PCR products of first amplification of HSP 70 (234) targeting analyzed by electrophoresis polyacrylamide gel stained with silver. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Infected Thrichomys laurentius from São Raimundo Nonato/PI; 2. Negative control of PCR reaction. (B) PCR products of re-amplification of the product obtained in the first PCR reaction. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Negative Thrichomys fosteri from Corumbá/MS (Positive only in kDNA); 2–5. Infected T. laurentius from São Raimundo Nonato/PI; 6. Infected T. fosteri from Corumbá/MS; 7. Negative control of PCR after re-amplification.
    Genelute Pcr Clean Up Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3166 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega wizard pcr preps dna purification system
    <t>UP-PCR</t> results of some patients. We have detected all the patients and finally we chose some positive bacteria and carried out the electrophoresis, so every line had PCR products. The deeper the stripe, the more the bacteria. Lines 1–9 represented PCR product of the 16S rRNA gene of Staphylococcus aureus , Staphylococcus epidermidis , Pseudomonas aeruginosa , Escherichia coli , Streptococcus viridans , Staphylococcus saprophyticus, Corynebacterium parvum , Klebsiella pneumoniae , and Enterobacter cloacae ; M for DL2000 <t>DNA</t> Marker; ten for negative control.
    Wizard Pcr Preps Dna Purification System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3010 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL pcr clean up
    Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific <t>PCR</t> analysis of the <t>chd</t> gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.
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    Qiagen ultraclean pcr clean up kit
    Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific <t>PCR</t> analysis of the <t>chd</t> gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.
    Ultraclean Pcr Clean Up Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1693 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen qiaquick pcr purification kit
    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic <t>DNA</t> purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using <t>PCR.</t> b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses
    Qiaquick Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 137129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr clean up kit
    Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by <t>PCR</t> using primers specific to these regions (a). L-100 bp ladder. PCR products were sequenced by Sangers method. All the 4 nucleotide differences were also present at genomic <t>DNA</t> level. Representative image of exon specific amplification (b). Alignment of exon specific sequencing results with human, mouse, and rat (predicted) sequence shows 2 amino acid residue changes (c).
    Pcr Clean Up Kit, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 1399 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa nucleospin gel
    Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the <t>NucleoSpin</t> Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.
    Nucleospin Gel, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 999 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Zymo Research dna clean and concentrator kit
    Effect of DAC concentration on <t>DNA</t> methylation and reactivation of p57 and p16 tumor suppressor genes . A), HL-60 cells were treated with the indicated concentrations of DAC for 48 h. Total DNA was isolated at 72 h and the LINE-1 element gene methylation status was analyzed by COBRA assay. B), HL-60 cells were treated with the indicated concentrations of DAC for 48 h and total RNA was isolated at 72 h. Gene expression of 18 S ribosomal RNA gene and p57 were analyzed by <t>RT-PCR.</t> The amount of DNA amplified during the exponential phase of PCR was analyzed by quantification of amplified DNA by an Agilent 2100 Bioanalyzer. The control cells are HL-60 with no drug treatment. Control vs DAC 100 ng/ml or 1,000 ng/ml p
    Dna Clean And Concentrator Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 1718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Zymo Research dna clean
    NGS analysis of phage integration sites. (A) A schematic diagram of NGS library preparation. Genomic <t>DNA</t> extracted from phage-infected E. coli (input DNA) is first enzymatically fragmented (step 1) and then ligated with adaptors (step 2). The fragments are <t>PCR</t> amplified with an adaptor-specific primer (gray) and a biotinylated primer specific to phage sequence (red) (PCR1) (step 3). The biotinylated PCR fragments are pulled down by magnetic streptavidin beads (step 4). Another round of PCR is performed using an adaptor-specific primer (gray) and phage-specific primer (red) containing the sequences necessary for Illumina sequencing. The phage-specific primer is internal to the primer used in PCR1, making this PCR seminested for increased specificity. (B) A genome-wide landscape of detected integration sites. On the outer ring, the positions of integration sites are shown. The color code indicates that the site was found in 1 (black), 2 to 4 (green), or > 4 (red) replicate samples. For those that were found in multiple (i.e., green and red) replicate samples, the names of the genes closest to each integration site are shown. *, sites that were also found in the work described in references 7 and 9 . The inner ring shows the motif scores of each integration site relative to the average score for all integration sites found. The dashed line indicates the average score of all possible 29-base genomic sequences. (C) A summary of the locations of the detected secondary integration sites. The secondary integration sites were categorized by strandness (left) or by their position with respect to host genes (top). The upstream and downstream positions are defined with respect to the gene closest to the integration site. (D) The sequence motif of integration sites. A total of 29 bases around each of the integration sites were inputted into the WebLogo program ( http://weblogo.berkeley.edu/ ) to generate the motif. For comparison, the corresponding attB sequence is also shown (top); bold letters indicate bases that match the motif.
    Dna Clean, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 3561 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa pcr clean up kit
    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain <t>DNA</t> (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time <t>RT-PCR</t> of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
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    Thermo Fisher purelink pcr micro kit
    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain <t>DNA</t> (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time <t>RT-PCR</t> of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
    Purelink Pcr Micro Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 988 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Zymo Research dna clean concentrator 5 kit
    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain <t>DNA</t> (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time <t>RT-PCR</t> of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
    Dna Clean Concentrator 5 Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 1280 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Journal: BMC Genomics

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    doi: 10.1186/s12864-017-4371-5

    Figure Lengend Snippet: DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Article Snippet: Similarly, DNAs were purified from de-crosslinked chromatin estimated to include 1 ng, 5 ng, 10 ng, and 50 ng of DNA after treatment of RNase A and proteinase K. The following reagents were used in the experiment: ChIP DNA Clean & Concentrator™ (Cat. # D5205) from Zymo Research (Zy) (Irvine, CA); Wizard® SV Gel and PCR Clean-Up System (Cat. # A9281) from Promega (Pr) (Fitchburg, WI); GeneJET PCR Purification Kit (Cat. # K0701) from Thermo Fisher Scientific (Th) (Waltham, MA); PureLink® PCR Purification Kit (Cat. # K310001) from Invitrogen (In) (Carlsbad, CA); Monarch® PCR & DNA Cleanup Kit (Cat. # T1030S) from New England Biolabs (Ne) (Ipswich, MA); Chromatin IP DNA Purification Kit (Cat. # 58002) from Active Motif (Am) (Carlsbad, CA); QIAquick PCR Purification Kit (Cat. # 28106) from Qiagen (Qp) (Valencia, CA), MinElute PCR Purification Kit (Cat. # 28006) from Qiagen (Qm); Agencourt AMPure XP (Cat. # A63881) from Beckman (Ba) (Indianapolis, IN), RNAClean™ XP (Cat. # A63987) from Beckman (Br), and phenol/chloroform extraction (PC) (Additional file ).

    Techniques: DNA Purification, Chromatin Immunoprecipitation, Purification, Generated, Derivative Assay, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    Screening and generation of Mp erf13 mutants (A) The flow chart of the screen for mutants defective in oil body formation from T-DNA-insertion lines. (B) A maximum likelihood phylogenetic tree of proteins with one ERF/AP2 domain. The color code is shown in Fig S1A . The branch lengths are proportional to the estimated number of substitutions per site. Bootstrap probability is indicated as a percentage on each branch with at least 50% support. A more detailed tree was presented previously ( 20 ). (C) Schematic representation of the Mp ERF13 gene structure and mutations generated in this study. Gray and black boxes indicate the UTR and coding sequences, respectively. Asterisks with numbers indicate sites of designed gRNA to generate Mp erf13-1 ge (*1 and *4) and Mp erf13-2 ge (*2 and *3). (D) PCR-based genotyping of Tak-1, Mp erf13 GOF , Mp erf13-1 ge , and Mp erf13-2 ge . The combinations and annealing sites of primers (a to d) are shown in (C). (E) The genomic and predicted amino acid sequences of the Mp ERF13 locus in Tak-1 and Mp erf13 mutants. Light blue, dark blue, and magenta letters indicate UTR, coding, and predicted amino acid sequences, respectively. The PAM sequences for gRNAs are underlined. The caret indicates the indel site. (F) Fluorescent and bright-field images of BODIPY-stained three-week-old thalli of Tak-1, Mp erf13-1 ge , and Mp erf13-2 ge . Bars = 0.5 mm. (G) The number of oil bodies visualized with BODIPY in a unit area (2.0 mm × 2.0 mm). Bars indicate means ± SD. Statistical analyses between Tak-1 and each genotype were conducted using a two-tailed Welch’s t -test. Sample numbers were 23 thalli for Tak-1, 24 for Mp erf13-1 ge , and 26 for Mp erf13-2 ge . p -values are 5.14×10 −11 for Mp erf13-1 ge and 5.14×10 −11 for Mp erf13-2 ge .

    Journal: bioRxiv

    Article Title: Switching secretory pathway direction for organelle acquisition in plants

    doi: 10.1101/2020.03.02.956961

    Figure Lengend Snippet: Screening and generation of Mp erf13 mutants (A) The flow chart of the screen for mutants defective in oil body formation from T-DNA-insertion lines. (B) A maximum likelihood phylogenetic tree of proteins with one ERF/AP2 domain. The color code is shown in Fig S1A . The branch lengths are proportional to the estimated number of substitutions per site. Bootstrap probability is indicated as a percentage on each branch with at least 50% support. A more detailed tree was presented previously ( 20 ). (C) Schematic representation of the Mp ERF13 gene structure and mutations generated in this study. Gray and black boxes indicate the UTR and coding sequences, respectively. Asterisks with numbers indicate sites of designed gRNA to generate Mp erf13-1 ge (*1 and *4) and Mp erf13-2 ge (*2 and *3). (D) PCR-based genotyping of Tak-1, Mp erf13 GOF , Mp erf13-1 ge , and Mp erf13-2 ge . The combinations and annealing sites of primers (a to d) are shown in (C). (E) The genomic and predicted amino acid sequences of the Mp ERF13 locus in Tak-1 and Mp erf13 mutants. Light blue, dark blue, and magenta letters indicate UTR, coding, and predicted amino acid sequences, respectively. The PAM sequences for gRNAs are underlined. The caret indicates the indel site. (F) Fluorescent and bright-field images of BODIPY-stained three-week-old thalli of Tak-1, Mp erf13-1 ge , and Mp erf13-2 ge . Bars = 0.5 mm. (G) The number of oil bodies visualized with BODIPY in a unit area (2.0 mm × 2.0 mm). Bars indicate means ± SD. Statistical analyses between Tak-1 and each genotype were conducted using a two-tailed Welch’s t -test. Sample numbers were 23 thalli for Tak-1, 24 for Mp erf13-1 ge , and 26 for Mp erf13-2 ge . p -values are 5.14×10 −11 for Mp erf13-1 ge and 5.14×10 −11 for Mp erf13-2 ge .

    Article Snippet: After agarose gel electrophoresis of the final TAIL-PCR products, DNA bands were excised and purified using the Wizard SV Gel and PCR Clean-Up System (Promega).

    Techniques: Generated, Polymerase Chain Reaction, Staining, Two Tailed Test

    Host and viral DNA content of differently treated ASFV samples. Quantitative dual PCR was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.

    Journal: Viruses

    Article Title: A Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever Virus

    doi: 10.3390/v11121129

    Figure Lengend Snippet: Host and viral DNA content of differently treated ASFV samples. Quantitative dual PCR was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.

    Article Snippet: Amplified DNA Clean Up REPLI-g samples were purified using the NucleoSpin Gel and PCR clean-up Kit (Macherey-Nagel Düren, Germany).

    Techniques: Polymerase Chain Reaction, Whole Genome Amplification

    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Journal: BMC Genomics

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    doi: 10.1186/s12864-017-4371-5

    Figure Lengend Snippet: DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Article Snippet: The Wizard® SV Gel and PCR Clean-Up System (Promega; Pr), the GeneJET PCR Purification Kit (Thermo Fisher Scientific; Th), the PureLink® PCR Purification Kit (Invitrogen; In) and the Chromatin IP DNA Purification Kit (Active Motif; Am) performed poorly with de-crosslinked chromatin.

    Techniques: DNA Purification, Chromatin Immunoprecipitation, Purification, Generated, Derivative Assay, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    Illustrative representation of HSP 70 (234) amplification for Leishmania sp. before and after re-amplification of the PCR products. (A) PCR products of first amplification of HSP 70 (234) targeting analyzed by electrophoresis polyacrylamide gel stained with silver. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Infected Thrichomys laurentius from São Raimundo Nonato/PI; 2. Negative control of PCR reaction. (B) PCR products of re-amplification of the product obtained in the first PCR reaction. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Negative Thrichomys fosteri from Corumbá/MS (Positive only in kDNA); 2–5. Infected T. laurentius from São Raimundo Nonato/PI; 6. Infected T. fosteri from Corumbá/MS; 7. Negative control of PCR after re-amplification.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Distinct Leishmania Species Infecting Wild Caviomorph Rodents (Rodentia: Hystricognathi) from Brazil

    doi: 10.1371/journal.pntd.0003389

    Figure Lengend Snippet: Illustrative representation of HSP 70 (234) amplification for Leishmania sp. before and after re-amplification of the PCR products. (A) PCR products of first amplification of HSP 70 (234) targeting analyzed by electrophoresis polyacrylamide gel stained with silver. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Infected Thrichomys laurentius from São Raimundo Nonato/PI; 2. Negative control of PCR reaction. (B) PCR products of re-amplification of the product obtained in the first PCR reaction. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Negative Thrichomys fosteri from Corumbá/MS (Positive only in kDNA); 2–5. Infected T. laurentius from São Raimundo Nonato/PI; 6. Infected T. fosteri from Corumbá/MS; 7. Negative control of PCR after re-amplification.

    Article Snippet: The PCR products obtained for HSP70 (234) targeting and the products positive only in kDNA targeting were purified using the Wizard SV Gel kit and PCR Clean-up System kit (Promega).

    Techniques: Amplification, Polymerase Chain Reaction, Electrophoresis, Staining, Molecular Weight, Marker, Infection, Negative Control, Mass Spectrometry

    High-throughput sequencing and mapping to α-satellite DNA reveals that centromeric CENP-A chromatin is an octameric nucleosome with transient DNA unwrapping of the DNA entry and exit sites at G1, G2, and mitosis. (A) Experimental design for obtaining CENP-A TAP – and H3.1 TAP -bound DNA sequences. (B) Microcapillary electrophoresis of MNase-digested bulk input mononucleosomes (top) and CENP-A TAP or H3.1 TAP or NH2 H3 CATD native ChIP (middle and bottom, immunoprecipitation). Purified CENP-A chromatin is nucleosome-like but protects a shorter DNA length than does H3.1-containing nucleosomes. (C) Quantitative real-time PCR for α-satellite DNA extracted from CENP-A TAP chromatin in random cycling (RC; magenta), G1 (red), and G2 (blue). n = 2 from two independent replicates. Error bars represent SEM. (D) CENP-A TAP – and H3.1 TAP -bound DNA was sequenced using paired-end 100-bp ChIP sequencing and then mapped to the human genome 38 assembly (hg38), which contains α-satellite sequence models for each centromere. Approximately 50% of CENP-A TAP –bound DNA is centromeric at G1 and G2. (E) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (F) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA on chromosome X were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (G) Distribution of the chromatin DNA lengths into two length bins: subnucleosomal (60–85 bp, as predicted for tetrasomes and hemisomes; Hasson et al., 2013 ) and nucleosomal (100–170 bp) for bulk input chromatin and affinity-purified CENP-A TAP and H3.1 TAP chromatin mapped to α-satellite DNA. n = 2 from two independent replicates. Error bars represent SEM.

    Journal: The Journal of Cell Biology

    Article Title: Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points

    doi: 10.1083/jcb.201608083

    Figure Lengend Snippet: High-throughput sequencing and mapping to α-satellite DNA reveals that centromeric CENP-A chromatin is an octameric nucleosome with transient DNA unwrapping of the DNA entry and exit sites at G1, G2, and mitosis. (A) Experimental design for obtaining CENP-A TAP – and H3.1 TAP -bound DNA sequences. (B) Microcapillary electrophoresis of MNase-digested bulk input mononucleosomes (top) and CENP-A TAP or H3.1 TAP or NH2 H3 CATD native ChIP (middle and bottom, immunoprecipitation). Purified CENP-A chromatin is nucleosome-like but protects a shorter DNA length than does H3.1-containing nucleosomes. (C) Quantitative real-time PCR for α-satellite DNA extracted from CENP-A TAP chromatin in random cycling (RC; magenta), G1 (red), and G2 (blue). n = 2 from two independent replicates. Error bars represent SEM. (D) CENP-A TAP – and H3.1 TAP -bound DNA was sequenced using paired-end 100-bp ChIP sequencing and then mapped to the human genome 38 assembly (hg38), which contains α-satellite sequence models for each centromere. Approximately 50% of CENP-A TAP –bound DNA is centromeric at G1 and G2. (E) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (F) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA on chromosome X were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (G) Distribution of the chromatin DNA lengths into two length bins: subnucleosomal (60–85 bp, as predicted for tetrasomes and hemisomes; Hasson et al., 2013 ) and nucleosomal (100–170 bp) for bulk input chromatin and affinity-purified CENP-A TAP and H3.1 TAP chromatin mapped to α-satellite DNA. n = 2 from two independent replicates. Error bars represent SEM.

    Article Snippet: DNA was purified from proteinase K–treated samples using a DNA purification kit following the manufacturer instructions (A9282; Promega) and was subsequently analyzed either by running a 2% low melting agarose (APEX) gel or by an Agilent Technologies 2100 Bioanalyzer by using the DNA 1000 kit.

    Techniques: Next-Generation Sequencing, Electrophoresis, Chromatin Immunoprecipitation, Immunoprecipitation, Purification, Real-time Polymerase Chain Reaction, Sequencing, Affinity Purification

    CENP-A chromatin has the physical characteristics of nucleosomes. (A) Experimental design for the separation of in vitro–reconstituted octameric nucleosomes or tetrasomes over a 5–25% sucrose gradient. (B) After sedimentation of in vitro–reconstituted H3 or CENP-A octameric nucleosomes or (CENP-A/H4) 2 or (H3/H4) 2 tetrasomes, fractions were immunoblotted for CENP-A or H3. (C) Experimental design for the sedimentation of in vivo bulk nucleosomes and short polynucleosomes at different points of the cell cycle. (D) Moderate MNase digestion profile of bulk chromatin from random cycling (RC) cells expressing CENP-A TAP or H3.1 TAP . (E) Sucrose gradient sedimentation and fractionation of bulk nucleosomes from CENP-A TAP –expressing cells synchronized at G1, G2, and mitosis. (top) Ethidium bromide stained DNA agarose gel revealing the DNA length extracted from the different fractions. (bottom) Immunoblot for CENP-A TAP . (F) Real-time quantitative PCR for α-satellite DNA in the different fractions (colored). Second axis shows quantification of CENP-A immunoblot shown in E. No CENP-A or α-satellite DNA was detected in fractions 7 and 9.

    Journal: The Journal of Cell Biology

    Article Title: Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points

    doi: 10.1083/jcb.201608083

    Figure Lengend Snippet: CENP-A chromatin has the physical characteristics of nucleosomes. (A) Experimental design for the separation of in vitro–reconstituted octameric nucleosomes or tetrasomes over a 5–25% sucrose gradient. (B) After sedimentation of in vitro–reconstituted H3 or CENP-A octameric nucleosomes or (CENP-A/H4) 2 or (H3/H4) 2 tetrasomes, fractions were immunoblotted for CENP-A or H3. (C) Experimental design for the sedimentation of in vivo bulk nucleosomes and short polynucleosomes at different points of the cell cycle. (D) Moderate MNase digestion profile of bulk chromatin from random cycling (RC) cells expressing CENP-A TAP or H3.1 TAP . (E) Sucrose gradient sedimentation and fractionation of bulk nucleosomes from CENP-A TAP –expressing cells synchronized at G1, G2, and mitosis. (top) Ethidium bromide stained DNA agarose gel revealing the DNA length extracted from the different fractions. (bottom) Immunoblot for CENP-A TAP . (F) Real-time quantitative PCR for α-satellite DNA in the different fractions (colored). Second axis shows quantification of CENP-A immunoblot shown in E. No CENP-A or α-satellite DNA was detected in fractions 7 and 9.

    Article Snippet: DNA was purified from proteinase K–treated samples using a DNA purification kit following the manufacturer instructions (A9282; Promega) and was subsequently analyzed either by running a 2% low melting agarose (APEX) gel or by an Agilent Technologies 2100 Bioanalyzer by using the DNA 1000 kit.

    Techniques: In Vitro, Sedimentation, In Vivo, Expressing, Fractionation, Staining, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

    UP-PCR results of some patients. We have detected all the patients and finally we chose some positive bacteria and carried out the electrophoresis, so every line had PCR products. The deeper the stripe, the more the bacteria. Lines 1–9 represented PCR product of the 16S rRNA gene of Staphylococcus aureus , Staphylococcus epidermidis , Pseudomonas aeruginosa , Escherichia coli , Streptococcus viridans , Staphylococcus saprophyticus, Corynebacterium parvum , Klebsiella pneumoniae , and Enterobacter cloacae ; M for DL2000 DNA Marker; ten for negative control.

    Journal: BioMed Research International

    Article Title: Genetic Identification and Risk Factor Analysis of Asymptomatic Bacterial Colonization on Cardiovascular Implantable Electronic Devices

    doi: 10.1155/2014/725163

    Figure Lengend Snippet: UP-PCR results of some patients. We have detected all the patients and finally we chose some positive bacteria and carried out the electrophoresis, so every line had PCR products. The deeper the stripe, the more the bacteria. Lines 1–9 represented PCR product of the 16S rRNA gene of Staphylococcus aureus , Staphylococcus epidermidis , Pseudomonas aeruginosa , Escherichia coli , Streptococcus viridans , Staphylococcus saprophyticus, Corynebacterium parvum , Klebsiella pneumoniae , and Enterobacter cloacae ; M for DL2000 DNA Marker; ten for negative control.

    Article Snippet: The PCR product was purified using Wizard PCR Preps DNA Purification System (Promega) and then ligated into the pGEM-T Easy Vector (Promega).

    Techniques: Polymerase Chain Reaction, Electrophoresis, Marker, Negative Control

    Predicted utility of the marker rescue approach to generate chimeric AAV vectors. The marker rescue approach can be utilized to generate chimeric virus from any combination of AAV serotypes. A capsid mutant of any serotype serves as the marker rescue plasmid template. (A) The mutation can be rescued via cotransfection capsid DNA from any other template. (B) After transfection of the cells with the mutant plasmid and capsid DNA, a mixed population of viral genomes is produced that can be PCR amplified and cloned into the shuttle vector, and individual clones can then be assessed for biological properties. (C and D) Alternatively, as a selection step the transfection lysate can be cycled onto any cell type of interest (C) or can be used to directly infect animals (D). (E) Ultimately, the cycling onto cells or in animals serves to enrich for a predominant viral recombinant.

    Journal: Journal of Virology

    Article Title: Marker Rescue of Adeno-Associated Virus (AAV) Capsid Mutants: a Novel Approach for Chimeric AAV Production

    doi: 10.1128/JVI.77.1.423-432.2003

    Figure Lengend Snippet: Predicted utility of the marker rescue approach to generate chimeric AAV vectors. The marker rescue approach can be utilized to generate chimeric virus from any combination of AAV serotypes. A capsid mutant of any serotype serves as the marker rescue plasmid template. (A) The mutation can be rescued via cotransfection capsid DNA from any other template. (B) After transfection of the cells with the mutant plasmid and capsid DNA, a mixed population of viral genomes is produced that can be PCR amplified and cloned into the shuttle vector, and individual clones can then be assessed for biological properties. (C and D) Alternatively, as a selection step the transfection lysate can be cycled onto any cell type of interest (C) or can be used to directly infect animals (D). (E) Ultimately, the cycling onto cells or in animals serves to enrich for a predominant viral recombinant.

    Article Snippet: The AAV3 PCR capsid fragments were digested with Dpn I to eliminate parental plasmid template and were purified with Wizard PCR Preps (Promega, Madison, Wis.) prior to transfection.

    Techniques: Marker, Mutagenesis, Plasmid Preparation, Cotransfection, Transfection, Produced, Polymerase Chain Reaction, Amplification, Clone Assay, Selection, Recombinant

    ) and contains a 12-bp in-frame linker (encoding the amino acids AISP) inserted at AAV2 nt 3761. The H/N3761 mutant assembles particles but is noninfectious due to a lack of heparin binding. (B) Marker rescue strategy to generate chimeric AAV. DNA from the three mutants (H/N3761, 1n532, and 1n562) was digested with Pvu II and cotransfected with the AAV3 PCR product. After the transfection, the 293 cells were coinfected with adenovirus dl 309. The lysates from the transfections (T) were applied in two successive cycles (C1 and C2) to HeLa cells and then infected with adenovirus dl 309. The resulting viruses were analyzed by dot blot, Western blot, PCR, and DNA sequencing analyses.

    Journal: Journal of Virology

    Article Title: Marker Rescue of Adeno-Associated Virus (AAV) Capsid Mutants: a Novel Approach for Chimeric AAV Production

    doi: 10.1128/JVI.77.1.423-432.2003

    Figure Lengend Snippet: ) and contains a 12-bp in-frame linker (encoding the amino acids AISP) inserted at AAV2 nt 3761. The H/N3761 mutant assembles particles but is noninfectious due to a lack of heparin binding. (B) Marker rescue strategy to generate chimeric AAV. DNA from the three mutants (H/N3761, 1n532, and 1n562) was digested with Pvu II and cotransfected with the AAV3 PCR product. After the transfection, the 293 cells were coinfected with adenovirus dl 309. The lysates from the transfections (T) were applied in two successive cycles (C1 and C2) to HeLa cells and then infected with adenovirus dl 309. The resulting viruses were analyzed by dot blot, Western blot, PCR, and DNA sequencing analyses.

    Article Snippet: The AAV3 PCR capsid fragments were digested with Dpn I to eliminate parental plasmid template and were purified with Wizard PCR Preps (Promega, Madison, Wis.) prior to transfection.

    Techniques: Mutagenesis, Binding Assay, Marker, Polymerase Chain Reaction, Transfection, Infection, Dot Blot, Western Blot, DNA Sequencing

    Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific PCR analysis of the chd gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.

    Journal: Scientific Reports

    Article Title: In vivo targeted single-nucleotide editing in zebrafish

    doi: 10.1038/s41598-018-29794-9

    Figure Lengend Snippet: Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific PCR analysis of the chd gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.

    Article Snippet: The PCR products from the chd and oep amplifications were purified with NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany) and sequenced by Fasmac (Atsugi, Japan) or by Macrogen Japan (Kyoto, Japan) with the chd _1st_F and oep _1st_F primers, respectively.

    Techniques: Fluorescence In Situ Hybridization, Polymerase Chain Reaction, Amplification, Sequencing

    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using PCR. b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses

    Journal: Epigenetics & Chromatin

    Article Title: Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination

    doi: 10.1186/s13072-018-0247-4

    Figure Lengend Snippet: TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using PCR. b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses

    Article Snippet: Immunoprecipitated bead–DNA complex was treated with proteinase K for 3 h at 50 °C in elution buffer and hydroxymethylated DNA purified by using the Qiagen PCR clean-up kit (Qiaquick).

    Techniques: Cell Culture, Purification, Immunoprecipitation, Polymerase Chain Reaction

    Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by PCR using primers specific to these regions (a). L-100 bp ladder. PCR products were sequenced by Sangers method. All the 4 nucleotide differences were also present at genomic DNA level. Representative image of exon specific amplification (b). Alignment of exon specific sequencing results with human, mouse, and rat (predicted) sequence shows 2 amino acid residue changes (c).

    Journal: BioMed Research International

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico

    doi: 10.1155/2017/5170680

    Figure Lengend Snippet: Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by PCR using primers specific to these regions (a). L-100 bp ladder. PCR products were sequenced by Sangers method. All the 4 nucleotide differences were also present at genomic DNA level. Representative image of exon specific amplification (b). Alignment of exon specific sequencing results with human, mouse, and rat (predicted) sequence shows 2 amino acid residue changes (c).

    Article Snippet: PCR amplified DNA was cleaned using PCR clean-up kit following the kit protocol (Promega, Wisconsin, USA), sequenced, and verified using nucleotide BLAST tool.

    Techniques: Amplification, Sequencing, Polymerase Chain Reaction

    Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the NucleoSpin Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.

    Journal: Frontiers in Microbiology

    Article Title: Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters

    doi: 10.3389/fmicb.2018.00830

    Figure Lengend Snippet: Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the NucleoSpin Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.

    Article Snippet: 10 μL) were purified using NucleoSpin Gel and the PCR Clean-up Kit (Takara Bio, Shiga, Japan).

    Techniques: Nested PCR, Amplification, Sequencing, Purification, Polymerase Chain Reaction, Chromatin Immunoprecipitation

    Effect of DAC concentration on DNA methylation and reactivation of p57 and p16 tumor suppressor genes . A), HL-60 cells were treated with the indicated concentrations of DAC for 48 h. Total DNA was isolated at 72 h and the LINE-1 element gene methylation status was analyzed by COBRA assay. B), HL-60 cells were treated with the indicated concentrations of DAC for 48 h and total RNA was isolated at 72 h. Gene expression of 18 S ribosomal RNA gene and p57 were analyzed by RT-PCR. The amount of DNA amplified during the exponential phase of PCR was analyzed by quantification of amplified DNA by an Agilent 2100 Bioanalyzer. The control cells are HL-60 with no drug treatment. Control vs DAC 100 ng/ml or 1,000 ng/ml p

    Journal: BMC Cancer

    Article Title: Importance of dose-schedule of 5-aza-2'-deoxycytidine for epigenetic therapy of cancer

    doi: 10.1186/1471-2407-8-128

    Figure Lengend Snippet: Effect of DAC concentration on DNA methylation and reactivation of p57 and p16 tumor suppressor genes . A), HL-60 cells were treated with the indicated concentrations of DAC for 48 h. Total DNA was isolated at 72 h and the LINE-1 element gene methylation status was analyzed by COBRA assay. B), HL-60 cells were treated with the indicated concentrations of DAC for 48 h and total RNA was isolated at 72 h. Gene expression of 18 S ribosomal RNA gene and p57 were analyzed by RT-PCR. The amount of DNA amplified during the exponential phase of PCR was analyzed by quantification of amplified DNA by an Agilent 2100 Bioanalyzer. The control cells are HL-60 with no drug treatment. Control vs DAC 100 ng/ml or 1,000 ng/ml p

    Article Snippet: The PCR products were then purified using the DNA Clean and Concentrator Kit (Zymo Research, CA, USA) and digested with Hinf I enzyme (New Englend BioLabs, MA, USA) according to manufacturer's protocols.

    Techniques: Concentration Assay, DNA Methylation Assay, Isolation, Methylation, Combined Bisulfite Restriction Analysis Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

    EMSA analysis indicates that the predicted SLII is important for DNA binding. (A) Nucleotide sequences of the pRSP pagA promoter region. The sequence shown is located immediately upstream of the translation start codon ATG (uppercase bold). The −35 and −10 regions of a σ A ). The hypothesized SLI is presented in bold and SLII is in bold italic. Transcription start site A is indicated in uppercase bold italic. (B) Map of PAG4-1 and PAG4-2 fragments within PAG4 in relation to the hypothesized stem-loops. Numbers indicate the distance from the transcriptional start site. The figure is drawn to scale. (C) EMSA on a 2% agarose EABE gel with AtxA WT incubated with PAG4 fragment and unlabeled competitor of either PAG4-1 or PAG4-2 generated via PCR. An excess of 10×, 30×, 60×, and 100× was added in increasing amounts to the EMSAs. (D) Map of RPAGΔSLI and RPAGΔSLII fragments in relation to the hypothesized stem-loops on the labeled RPAG700 fragment. Numbers indicate the distance from the transcriptional start site. The figure is drawn to scale. (E) EMSA on a 2% agarose EABE gel with AtxA WT incubated with the RPAG700 fragment and unlabeled competitor of either RPAGΔSLI or RPAGΔSLII generated via PCR. Excesses of 10×, 30×, and 60× were added in increasing amounts to the EMSA mixtures.

    Journal: Journal of Bacteriology

    Article Title: Bacillus anthracis Virulence Regulator AtxA Binds Specifically to the pagA Promoter Region

    doi: 10.1128/JB.00569-19

    Figure Lengend Snippet: EMSA analysis indicates that the predicted SLII is important for DNA binding. (A) Nucleotide sequences of the pRSP pagA promoter region. The sequence shown is located immediately upstream of the translation start codon ATG (uppercase bold). The −35 and −10 regions of a σ A ). The hypothesized SLI is presented in bold and SLII is in bold italic. Transcription start site A is indicated in uppercase bold italic. (B) Map of PAG4-1 and PAG4-2 fragments within PAG4 in relation to the hypothesized stem-loops. Numbers indicate the distance from the transcriptional start site. The figure is drawn to scale. (C) EMSA on a 2% agarose EABE gel with AtxA WT incubated with PAG4 fragment and unlabeled competitor of either PAG4-1 or PAG4-2 generated via PCR. An excess of 10×, 30×, 60×, and 100× was added in increasing amounts to the EMSAs. (D) Map of RPAGΔSLI and RPAGΔSLII fragments in relation to the hypothesized stem-loops on the labeled RPAG700 fragment. Numbers indicate the distance from the transcriptional start site. The figure is drawn to scale. (E) EMSA on a 2% agarose EABE gel with AtxA WT incubated with the RPAG700 fragment and unlabeled competitor of either RPAGΔSLI or RPAGΔSLII generated via PCR. Excesses of 10×, 30×, and 60× were added in increasing amounts to the EMSA mixtures.

    Article Snippet: Unlabeled PCR products were purified using the DNA Clean & Concentrator kit (Zymo Research, Irvine, CA), following the manufacturer’s protocol.

    Techniques: Binding Assay, Sequencing, Incubation, Generated, Polymerase Chain Reaction, Labeling

    NGS analysis of phage integration sites. (A) A schematic diagram of NGS library preparation. Genomic DNA extracted from phage-infected E. coli (input DNA) is first enzymatically fragmented (step 1) and then ligated with adaptors (step 2). The fragments are PCR amplified with an adaptor-specific primer (gray) and a biotinylated primer specific to phage sequence (red) (PCR1) (step 3). The biotinylated PCR fragments are pulled down by magnetic streptavidin beads (step 4). Another round of PCR is performed using an adaptor-specific primer (gray) and phage-specific primer (red) containing the sequences necessary for Illumina sequencing. The phage-specific primer is internal to the primer used in PCR1, making this PCR seminested for increased specificity. (B) A genome-wide landscape of detected integration sites. On the outer ring, the positions of integration sites are shown. The color code indicates that the site was found in 1 (black), 2 to 4 (green), or > 4 (red) replicate samples. For those that were found in multiple (i.e., green and red) replicate samples, the names of the genes closest to each integration site are shown. *, sites that were also found in the work described in references 7 and 9 . The inner ring shows the motif scores of each integration site relative to the average score for all integration sites found. The dashed line indicates the average score of all possible 29-base genomic sequences. (C) A summary of the locations of the detected secondary integration sites. The secondary integration sites were categorized by strandness (left) or by their position with respect to host genes (top). The upstream and downstream positions are defined with respect to the gene closest to the integration site. (D) The sequence motif of integration sites. A total of 29 bases around each of the integration sites were inputted into the WebLogo program ( http://weblogo.berkeley.edu/ ) to generate the motif. For comparison, the corresponding attB sequence is also shown (top); bold letters indicate bases that match the motif.

    Journal: mBio

    Article Title: Combining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology

    doi: 10.1128/mBio.01038-17

    Figure Lengend Snippet: NGS analysis of phage integration sites. (A) A schematic diagram of NGS library preparation. Genomic DNA extracted from phage-infected E. coli (input DNA) is first enzymatically fragmented (step 1) and then ligated with adaptors (step 2). The fragments are PCR amplified with an adaptor-specific primer (gray) and a biotinylated primer specific to phage sequence (red) (PCR1) (step 3). The biotinylated PCR fragments are pulled down by magnetic streptavidin beads (step 4). Another round of PCR is performed using an adaptor-specific primer (gray) and phage-specific primer (red) containing the sequences necessary for Illumina sequencing. The phage-specific primer is internal to the primer used in PCR1, making this PCR seminested for increased specificity. (B) A genome-wide landscape of detected integration sites. On the outer ring, the positions of integration sites are shown. The color code indicates that the site was found in 1 (black), 2 to 4 (green), or > 4 (red) replicate samples. For those that were found in multiple (i.e., green and red) replicate samples, the names of the genes closest to each integration site are shown. *, sites that were also found in the work described in references 7 and 9 . The inner ring shows the motif scores of each integration site relative to the average score for all integration sites found. The dashed line indicates the average score of all possible 29-base genomic sequences. (C) A summary of the locations of the detected secondary integration sites. The secondary integration sites were categorized by strandness (left) or by their position with respect to host genes (top). The upstream and downstream positions are defined with respect to the gene closest to the integration site. (D) The sequence motif of integration sites. A total of 29 bases around each of the integration sites were inputted into the WebLogo program ( http://weblogo.berkeley.edu/ ) to generate the motif. For comparison, the corresponding attB sequence is also shown (top); bold letters indicate bases that match the motif.

    Article Snippet: The PCR product was purified by the use of a DNA Clean and Concentrator-5 kit (Zymo) and then pulled down using Dynabeads MyOne streptavidin C1 magnetic beads (Thermo Fisher Scientific) per the manufacturer’s protocol.

    Techniques: Next-Generation Sequencing, Infection, Polymerase Chain Reaction, Amplification, Sequencing, Genome Wide, Genomic Sequencing

    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time RT-PCR of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition

    Journal: Nature Communications

    Article Title: Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice

    doi: 10.1038/s41467-019-13193-3

    Figure Lengend Snippet: Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time RT-PCR of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition

    Article Snippet: After linearization by digestion with DraI, the DNA fragment was purified with PCR Clean-up Kit (Clontech Laboratories) and transcribed using AmpliScribe T7-Flash Transcription Kit (Lucigen).

    Techniques: Immunofluorescence, Mouse Assay, Staining, Immunohistochemistry, Quantitative RT-PCR, Two Tailed Test