bcgi restriction enzymes  (New England Biolabs)


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    Name:
    BcgI
    Description:
    BcgI 1 250 units
    Catalog Number:
    r0545l
    Price:
    277
    Size:
    1 250 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs bcgi restriction enzymes
    BcgI
    BcgI 1 250 units
    https://www.bioz.com/result/bcgi restriction enzymes/product/New England Biolabs
    Average 92 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    bcgi restriction enzymes - by Bioz Stars, 2020-07
    92/100 stars

    Images

    1) Product Images from "Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation"

    Article Title: Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation

    Journal: Nature cell biology

    doi: 10.1038/ncb3209

    DNA-PK-phosphorylated FH promotes the DNA-PK complex accumulation at DSB regions and NHEJ a , Thymidine double block-synchronized U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were exposed to IR (10 Gy) and harvested 1 h after IR. Chromatin extracts or total cell lysates were prepared. b , DR-GFP-expressed U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were transfected with a vector with or without expressing I- SceI . ChIP analyses with antibodies that recognize FH (left panel) or Ku70 (right panel) and F1/R1 primers for the PCR were performed at the indicated time points after I- SceI transfection. The y-axis stands for the value of the I- SceI -induced fold increase of specific protein binding (the IP value was normalized to the input). The data represent the mean ± SD (n=3 independent experiments). c, d , DR-GFP-expressed U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were transfected with a vector with or without expressing I- SceI . PCR analyses for NHEJ were performed 42 h after transfection. A representative image of PCR products digested by I- SceI and BcgI is shown (left panel). The data represent the mean ± SD (n=3 independent experiments, right panel). * stands for P
    Figure Legend Snippet: DNA-PK-phosphorylated FH promotes the DNA-PK complex accumulation at DSB regions and NHEJ a , Thymidine double block-synchronized U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were exposed to IR (10 Gy) and harvested 1 h after IR. Chromatin extracts or total cell lysates were prepared. b , DR-GFP-expressed U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were transfected with a vector with or without expressing I- SceI . ChIP analyses with antibodies that recognize FH (left panel) or Ku70 (right panel) and F1/R1 primers for the PCR were performed at the indicated time points after I- SceI transfection. The y-axis stands for the value of the I- SceI -induced fold increase of specific protein binding (the IP value was normalized to the input). The data represent the mean ± SD (n=3 independent experiments). c, d , DR-GFP-expressed U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were transfected with a vector with or without expressing I- SceI . PCR analyses for NHEJ were performed 42 h after transfection. A representative image of PCR products digested by I- SceI and BcgI is shown (left panel). The data represent the mean ± SD (n=3 independent experiments, right panel). * stands for P

    Techniques Used: Non-Homologous End Joining, Blocking Assay, Expressing, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Protein Binding

    2) Product Images from "DNA Damage, Homology-Directed Repair, and DNA Methylation"

    Article Title: DNA Damage, Homology-Directed Repair, and DNA Methylation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030110

    DNA Methylation and Recombination (A) In the recombination assay primers for PCR and RT-PCR amplification were: (1) 5′-unrec (unrecombinant) centered on the I - SceI site, present only in cassette I and (2) 5′-rec (recombinant) centered in the BcgI site present only in the converted GFP or in cassette II. The 3′ end primer (3′) is located in cassette I in a sequence deleted in cassette II (X). The 5′-rec primer can pair with the 3′ primer only after its reconstitution in the 5′ cassette by gene conversion. The sequence of the 5′ primers is indicated with the distinguishing bases in capital letters. Stable Hela cells containing the DR-GFP plasmid were transiently transfected with I-SceI and pSVβGal (Promega) expression vectors as described in Materials and Methods. After 3 d, GFP cells were scored by FACS. The histogram shows data from three experiments. Transfection efficiency in these three experiments was 70% ± 5%. (B) Inhibition of methylation reveals silenced recombinants. Hela cells carrying the DR-GFP plasmid were transfected with I-SceI and PSVßGal expression vectors and treated with 5 μM 5-AzadC 48 h posttransfection, as described in Materials and Methods. (a) GFP + cells were analyzed by FACS. The ordinate shows the fraction of GFP + cells over total cells transfected. The efficiency of transfection was 70% +/− 5%. To determine the effect of 5-AzadC on GFP fluorescence, we compared the percentage of GFP + cells before and after 5-AzadC treatment in six experiments (three in duplicate) with a nonparametric matched pairs test, such as the Wilcoxon sign-rank ( p
    Figure Legend Snippet: DNA Methylation and Recombination (A) In the recombination assay primers for PCR and RT-PCR amplification were: (1) 5′-unrec (unrecombinant) centered on the I - SceI site, present only in cassette I and (2) 5′-rec (recombinant) centered in the BcgI site present only in the converted GFP or in cassette II. The 3′ end primer (3′) is located in cassette I in a sequence deleted in cassette II (X). The 5′-rec primer can pair with the 3′ primer only after its reconstitution in the 5′ cassette by gene conversion. The sequence of the 5′ primers is indicated with the distinguishing bases in capital letters. Stable Hela cells containing the DR-GFP plasmid were transiently transfected with I-SceI and pSVβGal (Promega) expression vectors as described in Materials and Methods. After 3 d, GFP cells were scored by FACS. The histogram shows data from three experiments. Transfection efficiency in these three experiments was 70% ± 5%. (B) Inhibition of methylation reveals silenced recombinants. Hela cells carrying the DR-GFP plasmid were transfected with I-SceI and PSVßGal expression vectors and treated with 5 μM 5-AzadC 48 h posttransfection, as described in Materials and Methods. (a) GFP + cells were analyzed by FACS. The ordinate shows the fraction of GFP + cells over total cells transfected. The efficiency of transfection was 70% +/− 5%. To determine the effect of 5-AzadC on GFP fluorescence, we compared the percentage of GFP + cells before and after 5-AzadC treatment in six experiments (three in duplicate) with a nonparametric matched pairs test, such as the Wilcoxon sign-rank ( p

    Techniques Used: DNA Methylation Assay, Recombination Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Recombinant, Sequencing, Plasmid Preparation, Transfection, Expressing, FACS, Inhibition, Methylation, Fluorescence

    Mapping of GFP Transcription in Recombinant and Nonrecombinant Cells with and without 5-AzadC Treatment (A) A schematic of the DR-GFP transcriptional unit shows the location of the CMV promoter, intron, and GFP coding sequence. Primers used for quantitative RT-PCR and RT-PCR are indicated by arrows. Different sets of primers were derived from the intron (738–757, forward 5′-CGTTACTCCCACAGGTGAGC-3′; 966–948, reverse 5′-CGCCCGTAGCGCTCACAGC-3′), AUG (1,666–1,685, forward 5′-TACAGCTCCTGGGCAACGTG-3′; 1,911–1,892, reverse, 5′-TCCTGCTCCTGGGCTTCTCG-3′), and BcgI/I-SceI (described in Figure 1 A) segments of GFP gene. Control (DR-GFP cells transfected with pBluescript), HR-L, and HR-H cells were treated with 40 μM 5-AzadC for 48 h. Total RNA was extracted 24 h later and subjected to quantitative RT-PCR with the primers indicated. The data, derived from three independent cDNAs, are shown as fold induction by 5-AzadC over the basal control, normalized to GADPH and β-actin. The primers used to amplify the control samples were those indicated as I-SceI unrec ( Figure 1 A). (B) Shown is RT-PCR with the same cDNAs indicated in (A) at 30 cycles.
    Figure Legend Snippet: Mapping of GFP Transcription in Recombinant and Nonrecombinant Cells with and without 5-AzadC Treatment (A) A schematic of the DR-GFP transcriptional unit shows the location of the CMV promoter, intron, and GFP coding sequence. Primers used for quantitative RT-PCR and RT-PCR are indicated by arrows. Different sets of primers were derived from the intron (738–757, forward 5′-CGTTACTCCCACAGGTGAGC-3′; 966–948, reverse 5′-CGCCCGTAGCGCTCACAGC-3′), AUG (1,666–1,685, forward 5′-TACAGCTCCTGGGCAACGTG-3′; 1,911–1,892, reverse, 5′-TCCTGCTCCTGGGCTTCTCG-3′), and BcgI/I-SceI (described in Figure 1 A) segments of GFP gene. Control (DR-GFP cells transfected with pBluescript), HR-L, and HR-H cells were treated with 40 μM 5-AzadC for 48 h. Total RNA was extracted 24 h later and subjected to quantitative RT-PCR with the primers indicated. The data, derived from three independent cDNAs, are shown as fold induction by 5-AzadC over the basal control, normalized to GADPH and β-actin. The primers used to amplify the control samples were those indicated as I-SceI unrec ( Figure 1 A). (B) Shown is RT-PCR with the same cDNAs indicated in (A) at 30 cycles.

    Techniques Used: Recombinant, Sequencing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Transfection

    Related Articles

    Transfection:

    Article Title: Cadherin Expression, Vectorial Active Transport, and Metallothionein Isoform 3 Mediated EMT/MET Responses in Cultured Primary and Immortalized Human Proximal Tubule Cells
    Article Snippet: .. The DNA constructs were linearized with BspH I and E-CDH ORF was linearized with Bcg I (New England BioLabs, Ipswich, MA) before transfection. .. The HK-2 cells were transfected using the Effectene transfection reagent (Qiagen, Valencia, CA) following the manufacturer’s protocol at a ratio of 1:10 plasmid to Effectene ratio, and the lipid complexes were added to the cells at 2 μg of DNA per 9.6 cm2 well of a 6-well culture plate.

    Article Title: DNA Damage, Homology-Directed Repair, and DNA Methylation
    Article Snippet: .. BcgI digestion of DNA or cDNA prior to PCR. cDNA prepared from 2 μg of total RNA extracted from Hela-DR-GFP transfected with pCASce or control plasmid was digested with BcgI restriction enzyme as recommended by the manufacturer (New England Biolabs, http://www.neb.com ). .. We used 2.5 μl of cleavage reaction to perform PCR with primers that specifically amplify unrecombinant (unrec) and recombinant (rec) GFP sequence.

    Amplification:

    Article Title: Nitric Oxide Dependent Downregulation of BRCA1 Expression Promotes Genetic Instability
    Article Snippet: .. After PCR amplification, a half-volume of PCR products was digested for 6 h at +37°C with 10 units of each of I-SceI and BcgI (NEB). ..

    Agarose Gel Electrophoresis:

    Article Title: Greater inotropic and cyclic AMP responses evoked by noradrenaline through Arg389 ?1-adrenoceptors versus Gly389 ?1-adrenoceptors in isolated human atrial myocardium
    Article Snippet: .. 10 μl of PCR product were then digested overnight at 37°C with 20 U of Eco 0109 I (New England Biolabs), 10 μl of BSA (100 μg ml−1 ), 2 μl of reaction buffer (49G > S) and similarly 2 U of Bcg I (New England Biolabs), 5 μl of 200 μ M S-adenylmethionine, 2 μl of supplied buffer (389G > R) before separation on a 1.5% agarose gel. ..

    Next-Generation Sequencing:

    Article Title: Population genetics analysis of the Nujiang catfish Creteuchiloglanis macropterus through a genome-wide single nucleotide polymorphisms resource generated by RAD-seq
    Article Snippet: .. The type IIB restriction enzyme chosen for the experiment was BcgI (New England BioLabs), which excises a 36-bp fragment around the recognition site, cleaves genomic DNA upstream and downstream of its target site, and generates tags of uniform length that are ideally suited for sequencing on existing NGS platforms. .. Digestion reactions were performed in a total volume of 6 µL, using 4 µL of intact, high-quality genomic DNA sample, each containing a total of 0.25 to 1 µg of DNA in 4 µL, 1 unit of BcgI, 0.6 µL of 10 × NEBuffer 3 and 0.4 µL of 150 µM SAM.

    Construct:

    Article Title: Cadherin Expression, Vectorial Active Transport, and Metallothionein Isoform 3 Mediated EMT/MET Responses in Cultured Primary and Immortalized Human Proximal Tubule Cells
    Article Snippet: .. The DNA constructs were linearized with BspH I and E-CDH ORF was linearized with Bcg I (New England BioLabs, Ipswich, MA) before transfection. .. The HK-2 cells were transfected using the Effectene transfection reagent (Qiagen, Valencia, CA) following the manufacturer’s protocol at a ratio of 1:10 plasmid to Effectene ratio, and the lipid complexes were added to the cells at 2 μg of DNA per 9.6 cm2 well of a 6-well culture plate.

    Polymerase Chain Reaction:

    Article Title: Greater inotropic and cyclic AMP responses evoked by noradrenaline through Arg389 ?1-adrenoceptors versus Gly389 ?1-adrenoceptors in isolated human atrial myocardium
    Article Snippet: .. 10 μl of PCR product were then digested overnight at 37°C with 20 U of Eco 0109 I (New England Biolabs), 10 μl of BSA (100 μg ml−1 ), 2 μl of reaction buffer (49G > S) and similarly 2 U of Bcg I (New England Biolabs), 5 μl of 200 μ M S-adenylmethionine, 2 μl of supplied buffer (389G > R) before separation on a 1.5% agarose gel. ..

    Article Title: DNA Damage, Homology-Directed Repair, and DNA Methylation
    Article Snippet: .. BcgI digestion of DNA or cDNA prior to PCR. cDNA prepared from 2 μg of total RNA extracted from Hela-DR-GFP transfected with pCASce or control plasmid was digested with BcgI restriction enzyme as recommended by the manufacturer (New England Biolabs, http://www.neb.com ). .. We used 2.5 μl of cleavage reaction to perform PCR with primers that specifically amplify unrecombinant (unrec) and recombinant (rec) GFP sequence.

    Article Title: Nitric Oxide Dependent Downregulation of BRCA1 Expression Promotes Genetic Instability
    Article Snippet: .. After PCR amplification, a half-volume of PCR products was digested for 6 h at +37°C with 10 units of each of I-SceI and BcgI (NEB). ..

    Generated:

    Article Title: ?1- and ?2c-adrenoreceptor variants as predictors of clinical aspects of dilated cardiomyopathy in people of African ancestry
    Article Snippet: .. BcgI (New England Biolabs) cleaves twice to excise its recognition site, accounting for the 34-bp discrepancy in the fragments generated. .. The restriction digests were electrophoresed on 3% agarose gels and visualised with ethidium bromide staining and ultraviolet illumination.

    Sequencing:

    Article Title: Population genetics analysis of the Nujiang catfish Creteuchiloglanis macropterus through a genome-wide single nucleotide polymorphisms resource generated by RAD-seq
    Article Snippet: .. The type IIB restriction enzyme chosen for the experiment was BcgI (New England BioLabs), which excises a 36-bp fragment around the recognition site, cleaves genomic DNA upstream and downstream of its target site, and generates tags of uniform length that are ideally suited for sequencing on existing NGS platforms. .. Digestion reactions were performed in a total volume of 6 µL, using 4 µL of intact, high-quality genomic DNA sample, each containing a total of 0.25 to 1 µg of DNA in 4 µL, 1 unit of BcgI, 0.6 µL of 10 × NEBuffer 3 and 0.4 µL of 150 µM SAM.

    Plasmid Preparation:

    Article Title: DNA Damage, Homology-Directed Repair, and DNA Methylation
    Article Snippet: .. BcgI digestion of DNA or cDNA prior to PCR. cDNA prepared from 2 μg of total RNA extracted from Hela-DR-GFP transfected with pCASce or control plasmid was digested with BcgI restriction enzyme as recommended by the manufacturer (New England Biolabs, http://www.neb.com ). .. We used 2.5 μl of cleavage reaction to perform PCR with primers that specifically amplify unrecombinant (unrec) and recombinant (rec) GFP sequence.

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    New England Biolabs bcgi restriction enzymes
    DNA-PK-phosphorylated FH promotes the DNA-PK complex accumulation at DSB regions and NHEJ a , Thymidine double block-synchronized U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were exposed to IR (10 Gy) and harvested 1 h after IR. Chromatin extracts or total cell lysates were prepared. b , DR-GFP-expressed U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were transfected with a vector with or without expressing I- <t>SceI</t> . ChIP analyses with antibodies that recognize FH (left panel) or Ku70 (right panel) and F1/R1 primers for the PCR were performed at the indicated time points after I- SceI transfection. The y-axis stands for the value of the I- SceI -induced fold increase of specific protein binding (the IP value was normalized to the input). The data represent the mean ± SD (n=3 independent experiments). c, d , DR-GFP-expressed U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were transfected with a vector with or without expressing I- SceI . PCR analyses for NHEJ were performed 42 h after transfection. A representative image of PCR products digested by I- SceI and <t>BcgI</t> is shown (left panel). The data represent the mean ± SD (n=3 independent experiments, right panel). * stands for P
    Bcgi Restriction Enzymes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bcgi restriction enzymes/product/New England Biolabs
    Average 92 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    bcgi restriction enzymes - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

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    DNA-PK-phosphorylated FH promotes the DNA-PK complex accumulation at DSB regions and NHEJ a , Thymidine double block-synchronized U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were exposed to IR (10 Gy) and harvested 1 h after IR. Chromatin extracts or total cell lysates were prepared. b , DR-GFP-expressed U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were transfected with a vector with or without expressing I- SceI . ChIP analyses with antibodies that recognize FH (left panel) or Ku70 (right panel) and F1/R1 primers for the PCR were performed at the indicated time points after I- SceI transfection. The y-axis stands for the value of the I- SceI -induced fold increase of specific protein binding (the IP value was normalized to the input). The data represent the mean ± SD (n=3 independent experiments). c, d , DR-GFP-expressed U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were transfected with a vector with or without expressing I- SceI . PCR analyses for NHEJ were performed 42 h after transfection. A representative image of PCR products digested by I- SceI and BcgI is shown (left panel). The data represent the mean ± SD (n=3 independent experiments, right panel). * stands for P

    Journal: Nature cell biology

    Article Title: Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation

    doi: 10.1038/ncb3209

    Figure Lengend Snippet: DNA-PK-phosphorylated FH promotes the DNA-PK complex accumulation at DSB regions and NHEJ a , Thymidine double block-synchronized U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were exposed to IR (10 Gy) and harvested 1 h after IR. Chromatin extracts or total cell lysates were prepared. b , DR-GFP-expressed U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were transfected with a vector with or without expressing I- SceI . ChIP analyses with antibodies that recognize FH (left panel) or Ku70 (right panel) and F1/R1 primers for the PCR were performed at the indicated time points after I- SceI transfection. The y-axis stands for the value of the I- SceI -induced fold increase of specific protein binding (the IP value was normalized to the input). The data represent the mean ± SD (n=3 independent experiments). c, d , DR-GFP-expressed U2OS cells with depleted endogenous FH and reconstituted expression of the indicated FH proteins were transfected with a vector with or without expressing I- SceI . PCR analyses for NHEJ were performed 42 h after transfection. A representative image of PCR products digested by I- SceI and BcgI is shown (left panel). The data represent the mean ± SD (n=3 independent experiments, right panel). * stands for P

    Article Snippet: I-SceI and BcgI restriction enzymes were purchased from New England Biolabs (Ipswich, MA).

    Techniques: Non-Homologous End Joining, Blocking Assay, Expressing, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Protein Binding

    DNA Methylation and Recombination (A) In the recombination assay primers for PCR and RT-PCR amplification were: (1) 5′-unrec (unrecombinant) centered on the I - SceI site, present only in cassette I and (2) 5′-rec (recombinant) centered in the BcgI site present only in the converted GFP or in cassette II. The 3′ end primer (3′) is located in cassette I in a sequence deleted in cassette II (X). The 5′-rec primer can pair with the 3′ primer only after its reconstitution in the 5′ cassette by gene conversion. The sequence of the 5′ primers is indicated with the distinguishing bases in capital letters. Stable Hela cells containing the DR-GFP plasmid were transiently transfected with I-SceI and pSVβGal (Promega) expression vectors as described in Materials and Methods. After 3 d, GFP cells were scored by FACS. The histogram shows data from three experiments. Transfection efficiency in these three experiments was 70% ± 5%. (B) Inhibition of methylation reveals silenced recombinants. Hela cells carrying the DR-GFP plasmid were transfected with I-SceI and PSVßGal expression vectors and treated with 5 μM 5-AzadC 48 h posttransfection, as described in Materials and Methods. (a) GFP + cells were analyzed by FACS. The ordinate shows the fraction of GFP + cells over total cells transfected. The efficiency of transfection was 70% +/− 5%. To determine the effect of 5-AzadC on GFP fluorescence, we compared the percentage of GFP + cells before and after 5-AzadC treatment in six experiments (three in duplicate) with a nonparametric matched pairs test, such as the Wilcoxon sign-rank ( p

    Journal: PLoS Genetics

    Article Title: DNA Damage, Homology-Directed Repair, and DNA Methylation

    doi: 10.1371/journal.pgen.0030110

    Figure Lengend Snippet: DNA Methylation and Recombination (A) In the recombination assay primers for PCR and RT-PCR amplification were: (1) 5′-unrec (unrecombinant) centered on the I - SceI site, present only in cassette I and (2) 5′-rec (recombinant) centered in the BcgI site present only in the converted GFP or in cassette II. The 3′ end primer (3′) is located in cassette I in a sequence deleted in cassette II (X). The 5′-rec primer can pair with the 3′ primer only after its reconstitution in the 5′ cassette by gene conversion. The sequence of the 5′ primers is indicated with the distinguishing bases in capital letters. Stable Hela cells containing the DR-GFP plasmid were transiently transfected with I-SceI and pSVβGal (Promega) expression vectors as described in Materials and Methods. After 3 d, GFP cells were scored by FACS. The histogram shows data from three experiments. Transfection efficiency in these three experiments was 70% ± 5%. (B) Inhibition of methylation reveals silenced recombinants. Hela cells carrying the DR-GFP plasmid were transfected with I-SceI and PSVßGal expression vectors and treated with 5 μM 5-AzadC 48 h posttransfection, as described in Materials and Methods. (a) GFP + cells were analyzed by FACS. The ordinate shows the fraction of GFP + cells over total cells transfected. The efficiency of transfection was 70% +/− 5%. To determine the effect of 5-AzadC on GFP fluorescence, we compared the percentage of GFP + cells before and after 5-AzadC treatment in six experiments (three in duplicate) with a nonparametric matched pairs test, such as the Wilcoxon sign-rank ( p

    Article Snippet: BcgI digestion of DNA or cDNA prior to PCR. cDNA prepared from 2 μg of total RNA extracted from Hela-DR-GFP transfected with pCASce or control plasmid was digested with BcgI restriction enzyme as recommended by the manufacturer (New England Biolabs, http://www.neb.com ).

    Techniques: DNA Methylation Assay, Recombination Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Recombinant, Sequencing, Plasmid Preparation, Transfection, Expressing, FACS, Inhibition, Methylation, Fluorescence

    Mapping of GFP Transcription in Recombinant and Nonrecombinant Cells with and without 5-AzadC Treatment (A) A schematic of the DR-GFP transcriptional unit shows the location of the CMV promoter, intron, and GFP coding sequence. Primers used for quantitative RT-PCR and RT-PCR are indicated by arrows. Different sets of primers were derived from the intron (738–757, forward 5′-CGTTACTCCCACAGGTGAGC-3′; 966–948, reverse 5′-CGCCCGTAGCGCTCACAGC-3′), AUG (1,666–1,685, forward 5′-TACAGCTCCTGGGCAACGTG-3′; 1,911–1,892, reverse, 5′-TCCTGCTCCTGGGCTTCTCG-3′), and BcgI/I-SceI (described in Figure 1 A) segments of GFP gene. Control (DR-GFP cells transfected with pBluescript), HR-L, and HR-H cells were treated with 40 μM 5-AzadC for 48 h. Total RNA was extracted 24 h later and subjected to quantitative RT-PCR with the primers indicated. The data, derived from three independent cDNAs, are shown as fold induction by 5-AzadC over the basal control, normalized to GADPH and β-actin. The primers used to amplify the control samples were those indicated as I-SceI unrec ( Figure 1 A). (B) Shown is RT-PCR with the same cDNAs indicated in (A) at 30 cycles.

    Journal: PLoS Genetics

    Article Title: DNA Damage, Homology-Directed Repair, and DNA Methylation

    doi: 10.1371/journal.pgen.0030110

    Figure Lengend Snippet: Mapping of GFP Transcription in Recombinant and Nonrecombinant Cells with and without 5-AzadC Treatment (A) A schematic of the DR-GFP transcriptional unit shows the location of the CMV promoter, intron, and GFP coding sequence. Primers used for quantitative RT-PCR and RT-PCR are indicated by arrows. Different sets of primers were derived from the intron (738–757, forward 5′-CGTTACTCCCACAGGTGAGC-3′; 966–948, reverse 5′-CGCCCGTAGCGCTCACAGC-3′), AUG (1,666–1,685, forward 5′-TACAGCTCCTGGGCAACGTG-3′; 1,911–1,892, reverse, 5′-TCCTGCTCCTGGGCTTCTCG-3′), and BcgI/I-SceI (described in Figure 1 A) segments of GFP gene. Control (DR-GFP cells transfected with pBluescript), HR-L, and HR-H cells were treated with 40 μM 5-AzadC for 48 h. Total RNA was extracted 24 h later and subjected to quantitative RT-PCR with the primers indicated. The data, derived from three independent cDNAs, are shown as fold induction by 5-AzadC over the basal control, normalized to GADPH and β-actin. The primers used to amplify the control samples were those indicated as I-SceI unrec ( Figure 1 A). (B) Shown is RT-PCR with the same cDNAs indicated in (A) at 30 cycles.

    Article Snippet: BcgI digestion of DNA or cDNA prior to PCR. cDNA prepared from 2 μg of total RNA extracted from Hela-DR-GFP transfected with pCASce or control plasmid was digested with BcgI restriction enzyme as recommended by the manufacturer (New England Biolabs, http://www.neb.com ).

    Techniques: Recombinant, Sequencing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Transfection