restriction enzyme bpuei  (New England Biolabs)


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    New England Biolabs restriction enzyme bpuei
    Confirmation of D2 −/− , I −/− and ID2 DKO cell lines. ( A ) <t>PCR</t> genotyping of D2 −/− and I −/− cells and targeting intermediates. Left panel : analyses of DNA fragments for WT (lane 1), D2 flox/Neo (lanes 2 and 3) and D2 −/− (lanes 4 and 5) after PCR amplification with primers FANCD2_EX11SF and FANCD2_LoxPSR flanking the targeted exon. Right panel : analyses of DNA fragments after PCR amplification from WT (lane 1), I flox/+ (lane 2) and I −/− (lanes 3 and 4) cells using primers FancIc_GG_LIF and FancIcond_GG_LoxR flanking the targeted exon. The PCR amplification spanning the targeted exons (exon 12 in FANCD2 ; exon 10 in FANCI ) was used to confirm the removal of the respective exon in the D2 −/− and I −/− cell lines. M: 1 kb markers. ( B ) Schematic of the FANCD2 targeting strategy in I −/− cells. CRISPR/Cas9-mediated gene targeting was used to functionally inactivate FANCD2 in the I −/− cell line. A guide RNA (purple sequence) was designed targeting FANCD2 exon 11 with the Cas9 cut site (red arrow) overlapping an endogenous <t>BpuEI</t> restriction enzyme recognition site (black bar). The PAM sequence of the sgRNA is shown in blue. Indels introduced at the Cas9 cut site should disrupt the BpuEI cleavage site. ( C ) Genotyping of ID2 DKO cells. PCR amplification and BpuEI restriction enzyme digestion of FANCD2 exon 11 in WT, I −/− and two ID2 −/− clones ( 1 and 2 ). Analyses of DNA fragments after PCR amplification with primers FancD2_CC_ F2 and FancD2_CC_ R2 (blue arrows from panel B) from WT (lanes 1 and 2), I −/− (lanes 3 and 4) and ID2 DKO cells (lanes 5, 6, 7 and 8) that had been untreated (lanes 1, 3, 5 and 7) or treated with BpuEI (lanes 2, 4, 6 and 8). Cleavage by BpuEI produces two faster migrating fragments (D, lanes 2 and 4). Resistance (R) to BpuEI digestion is seen in lanes 6 and 8 with the two ID2 DKO clones. ( D ) Sequence confirmation of CRISPR/Cas9 induced bi-allelic frameshift mutations in FANCD2 in the two ID2 DKO clones #1 and #2.
    Restriction Enzyme Bpuei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 1 article reviews
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    restriction enzyme bpuei - by Bioz Stars, 2020-04
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    1) Product Images from "FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response"

    Article Title: FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx847

    Confirmation of D2 −/− , I −/− and ID2 DKO cell lines. ( A ) PCR genotyping of D2 −/− and I −/− cells and targeting intermediates. Left panel : analyses of DNA fragments for WT (lane 1), D2 flox/Neo (lanes 2 and 3) and D2 −/− (lanes 4 and 5) after PCR amplification with primers FANCD2_EX11SF and FANCD2_LoxPSR flanking the targeted exon. Right panel : analyses of DNA fragments after PCR amplification from WT (lane 1), I flox/+ (lane 2) and I −/− (lanes 3 and 4) cells using primers FancIc_GG_LIF and FancIcond_GG_LoxR flanking the targeted exon. The PCR amplification spanning the targeted exons (exon 12 in FANCD2 ; exon 10 in FANCI ) was used to confirm the removal of the respective exon in the D2 −/− and I −/− cell lines. M: 1 kb markers. ( B ) Schematic of the FANCD2 targeting strategy in I −/− cells. CRISPR/Cas9-mediated gene targeting was used to functionally inactivate FANCD2 in the I −/− cell line. A guide RNA (purple sequence) was designed targeting FANCD2 exon 11 with the Cas9 cut site (red arrow) overlapping an endogenous BpuEI restriction enzyme recognition site (black bar). The PAM sequence of the sgRNA is shown in blue. Indels introduced at the Cas9 cut site should disrupt the BpuEI cleavage site. ( C ) Genotyping of ID2 DKO cells. PCR amplification and BpuEI restriction enzyme digestion of FANCD2 exon 11 in WT, I −/− and two ID2 −/− clones ( 1 and 2 ). Analyses of DNA fragments after PCR amplification with primers FancD2_CC_ F2 and FancD2_CC_ R2 (blue arrows from panel B) from WT (lanes 1 and 2), I −/− (lanes 3 and 4) and ID2 DKO cells (lanes 5, 6, 7 and 8) that had been untreated (lanes 1, 3, 5 and 7) or treated with BpuEI (lanes 2, 4, 6 and 8). Cleavage by BpuEI produces two faster migrating fragments (D, lanes 2 and 4). Resistance (R) to BpuEI digestion is seen in lanes 6 and 8 with the two ID2 DKO clones. ( D ) Sequence confirmation of CRISPR/Cas9 induced bi-allelic frameshift mutations in FANCD2 in the two ID2 DKO clones #1 and #2.
    Figure Legend Snippet: Confirmation of D2 −/− , I −/− and ID2 DKO cell lines. ( A ) PCR genotyping of D2 −/− and I −/− cells and targeting intermediates. Left panel : analyses of DNA fragments for WT (lane 1), D2 flox/Neo (lanes 2 and 3) and D2 −/− (lanes 4 and 5) after PCR amplification with primers FANCD2_EX11SF and FANCD2_LoxPSR flanking the targeted exon. Right panel : analyses of DNA fragments after PCR amplification from WT (lane 1), I flox/+ (lane 2) and I −/− (lanes 3 and 4) cells using primers FancIc_GG_LIF and FancIcond_GG_LoxR flanking the targeted exon. The PCR amplification spanning the targeted exons (exon 12 in FANCD2 ; exon 10 in FANCI ) was used to confirm the removal of the respective exon in the D2 −/− and I −/− cell lines. M: 1 kb markers. ( B ) Schematic of the FANCD2 targeting strategy in I −/− cells. CRISPR/Cas9-mediated gene targeting was used to functionally inactivate FANCD2 in the I −/− cell line. A guide RNA (purple sequence) was designed targeting FANCD2 exon 11 with the Cas9 cut site (red arrow) overlapping an endogenous BpuEI restriction enzyme recognition site (black bar). The PAM sequence of the sgRNA is shown in blue. Indels introduced at the Cas9 cut site should disrupt the BpuEI cleavage site. ( C ) Genotyping of ID2 DKO cells. PCR amplification and BpuEI restriction enzyme digestion of FANCD2 exon 11 in WT, I −/− and two ID2 −/− clones ( 1 and 2 ). Analyses of DNA fragments after PCR amplification with primers FancD2_CC_ F2 and FancD2_CC_ R2 (blue arrows from panel B) from WT (lanes 1 and 2), I −/− (lanes 3 and 4) and ID2 DKO cells (lanes 5, 6, 7 and 8) that had been untreated (lanes 1, 3, 5 and 7) or treated with BpuEI (lanes 2, 4, 6 and 8). Cleavage by BpuEI produces two faster migrating fragments (D, lanes 2 and 4). Resistance (R) to BpuEI digestion is seen in lanes 6 and 8 with the two ID2 DKO clones. ( D ) Sequence confirmation of CRISPR/Cas9 induced bi-allelic frameshift mutations in FANCD2 in the two ID2 DKO clones #1 and #2.

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

    Related Articles

    Clone Assay:

    Article Title: FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response
    Article Snippet: The gRNA was cloned into a CRISPR ( c lustered r egularly i nterspersed s hort p alindromic r epeats)/Cas9 plasmid (hSpCas9–2A-Puro/px459) as described ( ). .. Two days after transfection, the cells were subcloned, and individual subclones were screened for targeting by PCR amplification of exon 11 and by subsequent digestion with the restriction enzyme BpuEI (New England BioLabs, Inc.).

    Transfection:

    Article Title: FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response
    Article Snippet: .. Two days after transfection, the cells were subcloned, and individual subclones were screened for targeting by PCR amplification of exon 11 and by subsequent digestion with the restriction enzyme BpuEI (New England BioLabs, Inc.). .. Clones that were resistant to digestion with BpuEI were TOPO TA cloned (Life Technologies).

    Amplification:

    Article Title: FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response
    Article Snippet: .. Two days after transfection, the cells were subcloned, and individual subclones were screened for targeting by PCR amplification of exon 11 and by subsequent digestion with the restriction enzyme BpuEI (New England BioLabs, Inc.). .. Clones that were resistant to digestion with BpuEI were TOPO TA cloned (Life Technologies).

    Sample Prep:

    Article Title: GBStools: A Statistical Method for Estimating Allelic Dropout in Reduced Representation Sequencing Data
    Article Snippet: Library preparation Genomic DNA (50 ng) was digested with BpuEI (2.5 U), BsaXI (2 U), and CspCI (2.5 U) (NEB) at 37° for 90–120 min in buffer containing 20 μM S-adenosylmethionine. .. DNA end repair, 3' monoadenylation, and ligation of sequencing adapters were performed as described in the Illumina TruSeq DNA Sample Preparation Guide.

    Ligation:

    Article Title: GBStools: A Statistical Method for Estimating Allelic Dropout in Reduced Representation Sequencing Data
    Article Snippet: Library preparation Genomic DNA (50 ng) was digested with BpuEI (2.5 U), BsaXI (2 U), and CspCI (2.5 U) (NEB) at 37° for 90–120 min in buffer containing 20 μM S-adenosylmethionine. .. DNA end repair, 3' monoadenylation, and ligation of sequencing adapters were performed as described in the Illumina TruSeq DNA Sample Preparation Guide.

    Purification:

    Article Title: GBStools: A Statistical Method for Estimating Allelic Dropout in Reduced Representation Sequencing Data
    Article Snippet: Library preparation Genomic DNA (50 ng) was digested with BpuEI (2.5 U), BsaXI (2 U), and CspCI (2.5 U) (NEB) at 37° for 90–120 min in buffer containing 20 μM S-adenosylmethionine. .. The digestion product was purified on a DNA Clean and Concentrate column (Zymo Research).

    Sequencing:

    Article Title: GBStools: A Statistical Method for Estimating Allelic Dropout in Reduced Representation Sequencing Data
    Article Snippet: Library preparation Genomic DNA (50 ng) was digested with BpuEI (2.5 U), BsaXI (2 U), and CspCI (2.5 U) (NEB) at 37° for 90–120 min in buffer containing 20 μM S-adenosylmethionine. .. DNA end repair, 3' monoadenylation, and ligation of sequencing adapters were performed as described in the Illumina TruSeq DNA Sample Preparation Guide.

    Article Title: FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response
    Article Snippet: Two days after transfection, the cells were subcloned, and individual subclones were screened for targeting by PCR amplification of exon 11 and by subsequent digestion with the restriction enzyme BpuEI (New England BioLabs, Inc.). .. Sequencing of the TOPO TA clones confirmed targeted bi-allelic disruption that induced frameshifts in FANCD2 , generating an independent FANCD2 -null cell line, designated as clone #29 ( ).

    Derivative Assay:

    Article Title: GBStools: A Statistical Method for Estimating Allelic Dropout in Reduced Representation Sequencing Data
    Article Snippet: Library preparation Genomic DNA (50 ng) was digested with BpuEI (2.5 U), BsaXI (2 U), and CspCI (2.5 U) (NEB) at 37° for 90–120 min in buffer containing 20 μM S-adenosylmethionine. .. We designed a custom set of sequencing adapters, derived from the TruSeq adapters, with 65 six-bp barcodes ( ).

    CRISPR:

    Article Title: FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response
    Article Snippet: Paragraph title: CRISPR/Cas9 generation of FANCD2 −/− and FANCI−/− :FANCD2 −/− (ID2 DKO) cells ... Two days after transfection, the cells were subcloned, and individual subclones were screened for targeting by PCR amplification of exon 11 and by subsequent digestion with the restriction enzyme BpuEI (New England BioLabs, Inc.).

    Polymerase Chain Reaction:

    Article Title: FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response
    Article Snippet: .. Two days after transfection, the cells were subcloned, and individual subclones were screened for targeting by PCR amplification of exon 11 and by subsequent digestion with the restriction enzyme BpuEI (New England BioLabs, Inc.). .. Clones that were resistant to digestion with BpuEI were TOPO TA cloned (Life Technologies).

    Plasmid Preparation:

    Article Title: FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response
    Article Snippet: WT ( w ild- t ype) HCT116 cells were transfected with the CRISPR/Cas9 plasmid containing the gRNA targeting FANCD2 exon 11 using Lipofectamine 3000 (Life Technologies). .. Two days after transfection, the cells were subcloned, and individual subclones were screened for targeting by PCR amplification of exon 11 and by subsequent digestion with the restriction enzyme BpuEI (New England BioLabs, Inc.).

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    New England Biolabs restriction enzyme bpuei
    Confirmation of D2 −/− , I −/− and ID2 DKO cell lines. ( A ) <t>PCR</t> genotyping of D2 −/− and I −/− cells and targeting intermediates. Left panel : analyses of DNA fragments for WT (lane 1), D2 flox/Neo (lanes 2 and 3) and D2 −/− (lanes 4 and 5) after PCR amplification with primers FANCD2_EX11SF and FANCD2_LoxPSR flanking the targeted exon. Right panel : analyses of DNA fragments after PCR amplification from WT (lane 1), I flox/+ (lane 2) and I −/− (lanes 3 and 4) cells using primers FancIc_GG_LIF and FancIcond_GG_LoxR flanking the targeted exon. The PCR amplification spanning the targeted exons (exon 12 in FANCD2 ; exon 10 in FANCI ) was used to confirm the removal of the respective exon in the D2 −/− and I −/− cell lines. M: 1 kb markers. ( B ) Schematic of the FANCD2 targeting strategy in I −/− cells. CRISPR/Cas9-mediated gene targeting was used to functionally inactivate FANCD2 in the I −/− cell line. A guide RNA (purple sequence) was designed targeting FANCD2 exon 11 with the Cas9 cut site (red arrow) overlapping an endogenous <t>BpuEI</t> restriction enzyme recognition site (black bar). The PAM sequence of the sgRNA is shown in blue. Indels introduced at the Cas9 cut site should disrupt the BpuEI cleavage site. ( C ) Genotyping of ID2 DKO cells. PCR amplification and BpuEI restriction enzyme digestion of FANCD2 exon 11 in WT, I −/− and two ID2 −/− clones ( 1 and 2 ). Analyses of DNA fragments after PCR amplification with primers FancD2_CC_ F2 and FancD2_CC_ R2 (blue arrows from panel B) from WT (lanes 1 and 2), I −/− (lanes 3 and 4) and ID2 DKO cells (lanes 5, 6, 7 and 8) that had been untreated (lanes 1, 3, 5 and 7) or treated with BpuEI (lanes 2, 4, 6 and 8). Cleavage by BpuEI produces two faster migrating fragments (D, lanes 2 and 4). Resistance (R) to BpuEI digestion is seen in lanes 6 and 8 with the two ID2 DKO clones. ( D ) Sequence confirmation of CRISPR/Cas9 induced bi-allelic frameshift mutations in FANCD2 in the two ID2 DKO clones #1 and #2.
    Restriction Enzyme Bpuei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction enzyme bpuei/product/New England Biolabs
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    restriction enzyme bpuei - by Bioz Stars, 2020-04
    85/100 stars
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    Confirmation of D2 −/− , I −/− and ID2 DKO cell lines. ( A ) PCR genotyping of D2 −/− and I −/− cells and targeting intermediates. Left panel : analyses of DNA fragments for WT (lane 1), D2 flox/Neo (lanes 2 and 3) and D2 −/− (lanes 4 and 5) after PCR amplification with primers FANCD2_EX11SF and FANCD2_LoxPSR flanking the targeted exon. Right panel : analyses of DNA fragments after PCR amplification from WT (lane 1), I flox/+ (lane 2) and I −/− (lanes 3 and 4) cells using primers FancIc_GG_LIF and FancIcond_GG_LoxR flanking the targeted exon. The PCR amplification spanning the targeted exons (exon 12 in FANCD2 ; exon 10 in FANCI ) was used to confirm the removal of the respective exon in the D2 −/− and I −/− cell lines. M: 1 kb markers. ( B ) Schematic of the FANCD2 targeting strategy in I −/− cells. CRISPR/Cas9-mediated gene targeting was used to functionally inactivate FANCD2 in the I −/− cell line. A guide RNA (purple sequence) was designed targeting FANCD2 exon 11 with the Cas9 cut site (red arrow) overlapping an endogenous BpuEI restriction enzyme recognition site (black bar). The PAM sequence of the sgRNA is shown in blue. Indels introduced at the Cas9 cut site should disrupt the BpuEI cleavage site. ( C ) Genotyping of ID2 DKO cells. PCR amplification and BpuEI restriction enzyme digestion of FANCD2 exon 11 in WT, I −/− and two ID2 −/− clones ( 1 and 2 ). Analyses of DNA fragments after PCR amplification with primers FancD2_CC_ F2 and FancD2_CC_ R2 (blue arrows from panel B) from WT (lanes 1 and 2), I −/− (lanes 3 and 4) and ID2 DKO cells (lanes 5, 6, 7 and 8) that had been untreated (lanes 1, 3, 5 and 7) or treated with BpuEI (lanes 2, 4, 6 and 8). Cleavage by BpuEI produces two faster migrating fragments (D, lanes 2 and 4). Resistance (R) to BpuEI digestion is seen in lanes 6 and 8 with the two ID2 DKO clones. ( D ) Sequence confirmation of CRISPR/Cas9 induced bi-allelic frameshift mutations in FANCD2 in the two ID2 DKO clones #1 and #2.

    Journal: Nucleic Acids Research

    Article Title: FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response

    doi: 10.1093/nar/gkx847

    Figure Lengend Snippet: Confirmation of D2 −/− , I −/− and ID2 DKO cell lines. ( A ) PCR genotyping of D2 −/− and I −/− cells and targeting intermediates. Left panel : analyses of DNA fragments for WT (lane 1), D2 flox/Neo (lanes 2 and 3) and D2 −/− (lanes 4 and 5) after PCR amplification with primers FANCD2_EX11SF and FANCD2_LoxPSR flanking the targeted exon. Right panel : analyses of DNA fragments after PCR amplification from WT (lane 1), I flox/+ (lane 2) and I −/− (lanes 3 and 4) cells using primers FancIc_GG_LIF and FancIcond_GG_LoxR flanking the targeted exon. The PCR amplification spanning the targeted exons (exon 12 in FANCD2 ; exon 10 in FANCI ) was used to confirm the removal of the respective exon in the D2 −/− and I −/− cell lines. M: 1 kb markers. ( B ) Schematic of the FANCD2 targeting strategy in I −/− cells. CRISPR/Cas9-mediated gene targeting was used to functionally inactivate FANCD2 in the I −/− cell line. A guide RNA (purple sequence) was designed targeting FANCD2 exon 11 with the Cas9 cut site (red arrow) overlapping an endogenous BpuEI restriction enzyme recognition site (black bar). The PAM sequence of the sgRNA is shown in blue. Indels introduced at the Cas9 cut site should disrupt the BpuEI cleavage site. ( C ) Genotyping of ID2 DKO cells. PCR amplification and BpuEI restriction enzyme digestion of FANCD2 exon 11 in WT, I −/− and two ID2 −/− clones ( 1 and 2 ). Analyses of DNA fragments after PCR amplification with primers FancD2_CC_ F2 and FancD2_CC_ R2 (blue arrows from panel B) from WT (lanes 1 and 2), I −/− (lanes 3 and 4) and ID2 DKO cells (lanes 5, 6, 7 and 8) that had been untreated (lanes 1, 3, 5 and 7) or treated with BpuEI (lanes 2, 4, 6 and 8). Cleavage by BpuEI produces two faster migrating fragments (D, lanes 2 and 4). Resistance (R) to BpuEI digestion is seen in lanes 6 and 8 with the two ID2 DKO clones. ( D ) Sequence confirmation of CRISPR/Cas9 induced bi-allelic frameshift mutations in FANCD2 in the two ID2 DKO clones #1 and #2.

    Article Snippet: Two days after transfection, the cells were subcloned, and individual subclones were screened for targeting by PCR amplification of exon 11 and by subsequent digestion with the restriction enzyme BpuEI (New England BioLabs, Inc.).

    Techniques: Polymerase Chain Reaction, Amplification, CRISPR, Sequencing, Clone Assay