Structured Review

Axygen pcr cleanup kit
Phenotypes of the pre6 mutants and the PRE6 overexpression plants. (A) A diagram showing the <t>T-DNA</t> insertion sites in the pre6-1 and pre6-2 mutants. (B) Expression of PRE6 transcript in the pre6 mutants ( pre6-1 and pre6-2 ) and transgenic plants overexpressing PRE6 ( 35S:PRE6-1 and 35S:PRE6-2 ). RNA was isolated from 14-day-old seedlings, and <t>RT-PCR</t> was used to examine the expression of PRE6 . The expression of ACT2 was used as a control. (C) Photographs of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Sterilized seeds were grown on vertically placed 1/2 MS plates in darkness, and photographs were taken under a microscope. (D) Hypocotyl length of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Photographs were taken under a microscope, and ImageJ software was used to measure the hypocotyl length. Data represent the mean ± SD of 33–35 seedlings. ∗ Significantly different from the Col wild type seedlings ( p
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Images

1) Product Images from "Involvement of PACLOBUTRAZOL RESISTANCE6/KIDARI, an Atypical bHLH Transcription Factor, in Auxin Responses in Arabidopsis"

Article Title: Involvement of PACLOBUTRAZOL RESISTANCE6/KIDARI, an Atypical bHLH Transcription Factor, in Auxin Responses in Arabidopsis

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2017.01813

Phenotypes of the pre6 mutants and the PRE6 overexpression plants. (A) A diagram showing the T-DNA insertion sites in the pre6-1 and pre6-2 mutants. (B) Expression of PRE6 transcript in the pre6 mutants ( pre6-1 and pre6-2 ) and transgenic plants overexpressing PRE6 ( 35S:PRE6-1 and 35S:PRE6-2 ). RNA was isolated from 14-day-old seedlings, and RT-PCR was used to examine the expression of PRE6 . The expression of ACT2 was used as a control. (C) Photographs of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Sterilized seeds were grown on vertically placed 1/2 MS plates in darkness, and photographs were taken under a microscope. (D) Hypocotyl length of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Photographs were taken under a microscope, and ImageJ software was used to measure the hypocotyl length. Data represent the mean ± SD of 33–35 seedlings. ∗ Significantly different from the Col wild type seedlings ( p
Figure Legend Snippet: Phenotypes of the pre6 mutants and the PRE6 overexpression plants. (A) A diagram showing the T-DNA insertion sites in the pre6-1 and pre6-2 mutants. (B) Expression of PRE6 transcript in the pre6 mutants ( pre6-1 and pre6-2 ) and transgenic plants overexpressing PRE6 ( 35S:PRE6-1 and 35S:PRE6-2 ). RNA was isolated from 14-day-old seedlings, and RT-PCR was used to examine the expression of PRE6 . The expression of ACT2 was used as a control. (C) Photographs of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Sterilized seeds were grown on vertically placed 1/2 MS plates in darkness, and photographs were taken under a microscope. (D) Hypocotyl length of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Photographs were taken under a microscope, and ImageJ software was used to measure the hypocotyl length. Data represent the mean ± SD of 33–35 seedlings. ∗ Significantly different from the Col wild type seedlings ( p

Techniques Used: Over Expression, Expressing, Transgenic Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Mass Spectrometry, Microscopy, Software

2) Product Images from "Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep"

Article Title: Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep

Journal: Scientific Reports

doi: 10.1038/srep32271

Evaluation of sgRNA:Cas9-mediated genetic modifications in lambs. ( a ) PCR products of the targeted region of the MSTN , ASIP , and BCO2 genes of founder sheep co-microinjected with a mixture of Cas9 mRNA and sgRNAs. D2000 DNA Marker was used as a marker reference. ( b ) Detection of sgRNA:Cas9-mediated on-target cleavage of the MSTN , ASIP and BCO2 genes by using a T7EI cleavage assay. All PCR products from ( a ) were subjected to the T7EI cleavage assay. ( c ) Sequencing results of the modified MSTN , ASIP and BCO2 loci that were detected in the founder animals. Target sequences complementary to sgRNAs of targeted genes are in red text, while the PAM sequences are marked in green. The mutations are marked in blue, dashlines indicates deletions, and lowercase indicates insertions or replacements. Insertions (+), deletions (−), mutations (m) is shown to the right of each allele. The genotypes are shown to the right with the rates of total clones for TA-sequencing.
Figure Legend Snippet: Evaluation of sgRNA:Cas9-mediated genetic modifications in lambs. ( a ) PCR products of the targeted region of the MSTN , ASIP , and BCO2 genes of founder sheep co-microinjected with a mixture of Cas9 mRNA and sgRNAs. D2000 DNA Marker was used as a marker reference. ( b ) Detection of sgRNA:Cas9-mediated on-target cleavage of the MSTN , ASIP and BCO2 genes by using a T7EI cleavage assay. All PCR products from ( a ) were subjected to the T7EI cleavage assay. ( c ) Sequencing results of the modified MSTN , ASIP and BCO2 loci that were detected in the founder animals. Target sequences complementary to sgRNAs of targeted genes are in red text, while the PAM sequences are marked in green. The mutations are marked in blue, dashlines indicates deletions, and lowercase indicates insertions or replacements. Insertions (+), deletions (−), mutations (m) is shown to the right of each allele. The genotypes are shown to the right with the rates of total clones for TA-sequencing.

Techniques Used: Polymerase Chain Reaction, Marker, Cleavage Assay, Sequencing, Modification, Clone Assay

3) Product Images from "CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients"

Article Title: CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients

Journal: Scientific Reports

doi: 10.1038/srep20070

Cas9 mediated efficient hPD-1 KO in primary human T cells of healthy donors and patients. Freshly isolated PBMC were activated in vitro by IFN-γ for 3 d and IL-2 and aCD3 for 2 d and were transfected with pST1374-Cas9-GFP and pGL3-U6-hPD-1-sgRNA plasmids for each reaction. Sample G and Z stand for two individual patients and H stands for a healthy donor. ( a ) The GFP expression was evaluated by fluorescence microscope 24 h after electroporation (Donor 1 and Donor 2 are patients. Donor 3 is a representative of healthy donor). ( b ) PCR products were amplified and subjected to T7EN1 cleavage assay. Samples with cleavage bands were marked with an asterisk “*”. Sample G1/H1/Z1 represent control T cells and G2/H2/Z2 represent hPD-1 KO T cells. “ + ” represents for the positive control with the cleavage bands detected on HeLa cell line. NC, negative control. ( c ) DNA sequences of marked samples. TA clones from the PCR products were analyzed by DNA sequencing. The PAM sequences are underlined and highlighted in green; the targeting sequences in red; the mutations in blue, lower case; deletions (−), and insertions (+). The above experiments have been repeated 3 times with similar results.
Figure Legend Snippet: Cas9 mediated efficient hPD-1 KO in primary human T cells of healthy donors and patients. Freshly isolated PBMC were activated in vitro by IFN-γ for 3 d and IL-2 and aCD3 for 2 d and were transfected with pST1374-Cas9-GFP and pGL3-U6-hPD-1-sgRNA plasmids for each reaction. Sample G and Z stand for two individual patients and H stands for a healthy donor. ( a ) The GFP expression was evaluated by fluorescence microscope 24 h after electroporation (Donor 1 and Donor 2 are patients. Donor 3 is a representative of healthy donor). ( b ) PCR products were amplified and subjected to T7EN1 cleavage assay. Samples with cleavage bands were marked with an asterisk “*”. Sample G1/H1/Z1 represent control T cells and G2/H2/Z2 represent hPD-1 KO T cells. “ + ” represents for the positive control with the cleavage bands detected on HeLa cell line. NC, negative control. ( c ) DNA sequences of marked samples. TA clones from the PCR products were analyzed by DNA sequencing. The PAM sequences are underlined and highlighted in green; the targeting sequences in red; the mutations in blue, lower case; deletions (−), and insertions (+). The above experiments have been repeated 3 times with similar results.

Techniques Used: Isolation, In Vitro, Transfection, Expressing, Fluorescence, Microscopy, Electroporation, Polymerase Chain Reaction, Amplification, Cleavage Assay, Positive Control, Negative Control, Clone Assay, DNA Sequencing

Evaluation of hPD-1 sgRNA:Cas9-mediated modifications of human PD-1. ( a ) Schematic diagram of sgRNAs targeting at hPD-1 Exon 2 locus. The sgRNAs targeting sites on the sense strand are colored with blue while those on the antisense are colored with red. PAM sequences are underlined. ( b ) Detection of sgRNA:Cas9-mediated cleavage of hPD-1 by PCR and T7EN1 cleavage assay. M, DNA marker. sg1, sgRNA 1; sg2, sgRNA 2; sg3, sgRNA 3; sg4, sgRNA 4; sg (1 + 2), sgRNA 1 combined with sgRNA 2; sg (3 + 4), sgRNA 3 combined with sgRNA 4. Con, negative control. The above experiments have been repeated 3 times with similar results.
Figure Legend Snippet: Evaluation of hPD-1 sgRNA:Cas9-mediated modifications of human PD-1. ( a ) Schematic diagram of sgRNAs targeting at hPD-1 Exon 2 locus. The sgRNAs targeting sites on the sense strand are colored with blue while those on the antisense are colored with red. PAM sequences are underlined. ( b ) Detection of sgRNA:Cas9-mediated cleavage of hPD-1 by PCR and T7EN1 cleavage assay. M, DNA marker. sg1, sgRNA 1; sg2, sgRNA 2; sg3, sgRNA 3; sg4, sgRNA 4; sg (1 + 2), sgRNA 1 combined with sgRNA 2; sg (3 + 4), sgRNA 3 combined with sgRNA 4. Con, negative control. The above experiments have been repeated 3 times with similar results.

Techniques Used: Polymerase Chain Reaction, Cleavage Assay, Marker, Negative Control

4) Product Images from "Efficient Knockin Mouse Generation by ssDNA Oligonucleotides and Zinc-Finger Nuclease Assisted Homologous Recombination in Zygotes"

Article Title: Efficient Knockin Mouse Generation by ssDNA Oligonucleotides and Zinc-Finger Nuclease Assisted Homologous Recombination in Zygotes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0077696

Generation of ZFN-mediated c-kit mutant mice. a. Schematic showing the layout of ZFN target sites. The left and right ZFN binding sites with the spacer region are shown. b. Sequence analysis of the modified c-kit allele in 44 founder animals. The targeted region on the mouse c-kit locus was PCR-amplified from genomic DNA extracted from tails of the 5-day-old F0 pups, then subjected to T-A cloning. Colonies were randomly picked for DNA sequencing. Indels and mutations were detected in 8 out of 27 founders treated with 2.5 ng/μl ZFN mRNA and 7 out of 17 founders treated with 5 ng/μl ZFN mRNA. The ZFN binding site is underlined. Indels and mutations are highlighted in red.
Figure Legend Snippet: Generation of ZFN-mediated c-kit mutant mice. a. Schematic showing the layout of ZFN target sites. The left and right ZFN binding sites with the spacer region are shown. b. Sequence analysis of the modified c-kit allele in 44 founder animals. The targeted region on the mouse c-kit locus was PCR-amplified from genomic DNA extracted from tails of the 5-day-old F0 pups, then subjected to T-A cloning. Colonies were randomly picked for DNA sequencing. Indels and mutations were detected in 8 out of 27 founders treated with 2.5 ng/μl ZFN mRNA and 7 out of 17 founders treated with 5 ng/μl ZFN mRNA. The ZFN binding site is underlined. Indels and mutations are highlighted in red.

Techniques Used: Mutagenesis, Mouse Assay, Binding Assay, Sequencing, Modification, Polymerase Chain Reaction, Amplification, Clone Assay, DNA Sequencing

Targeted integration of a loxP site using ZFNc-kit and gene-targeting ssODNs . a. A schematic of the mouse c-kit locus and the ssODN sequences used to introduce a loxP site (in blue) into the genome in vivo . b. The representative gel electropherogram of the genotyping results by PCR amplification. 5 day old pups were genotyped by PCR amplification of tail genomic DNA. The expected wild type band is 178 bp (WT) and the expected mutant band is 214 bp. The PCR primers are listed in Table S1 . Ng, negative control. c. Sequence analysis of the mutant c-kit allele of the 9 founders. The target region of the mouse c-kit gene was PCR-amplified from tail genomic DNA of the 5-day-old founders and subjected to T-A cloning. Colonies were randomly picked for DNA sequencing. Precise loxP site insertion was detected in 2 out of 9 founders. ZFN binding site is underlined. The loxP sites are highlighted in blue. Indels are highlighted in red. WT, wild type.
Figure Legend Snippet: Targeted integration of a loxP site using ZFNc-kit and gene-targeting ssODNs . a. A schematic of the mouse c-kit locus and the ssODN sequences used to introduce a loxP site (in blue) into the genome in vivo . b. The representative gel electropherogram of the genotyping results by PCR amplification. 5 day old pups were genotyped by PCR amplification of tail genomic DNA. The expected wild type band is 178 bp (WT) and the expected mutant band is 214 bp. The PCR primers are listed in Table S1 . Ng, negative control. c. Sequence analysis of the mutant c-kit allele of the 9 founders. The target region of the mouse c-kit gene was PCR-amplified from tail genomic DNA of the 5-day-old founders and subjected to T-A cloning. Colonies were randomly picked for DNA sequencing. Precise loxP site insertion was detected in 2 out of 9 founders. ZFN binding site is underlined. The loxP sites are highlighted in blue. Indels are highlighted in red. WT, wild type.

Techniques Used: Introduce, In Vivo, Polymerase Chain Reaction, Amplification, Mutagenesis, Negative Control, Sequencing, Clone Assay, DNA Sequencing, Binding Assay

Targeted insertion of a restriction site using ZFNc-kit and gene-targeting ssODNs. a. A schematic of the mouse c-kit locus and the ssODN sequences of different sizes designed to introduce an exogenous BglII restriction site (in blue) into the genome in vivo using ZFNc-kit and ssODNs. b. Detection of introduced BglII site in founder animals. The representative gel electropherogram of genotyping results by BglII digestion of PCR product. F0 pups were genotyped by BglII digestion of the 592 bp PCR product amplified from tail genomic DNA. The PCR product from the wild type c-kit allele cannot be digested by BglII (WT), while the PCR product from the targeted mutant allele (10 out of 109 founders) can be digested by BglII into two distinct fragments (238 bp and 354 bp). The PCR primers are listed in Table S1 . c. Sequence analysis of the c-kit mutant allele of the 10 founders with correct BglII restriction bands. The target region of the mouse c-kit gene was PCR-amplified from tail genomic DNA of the 5-day-old founders and subjected to T-A cloning. Colonies were randomly picked for DNA sequencing. Precise BglII site insertion was detected in 8 out of 10 mutant founders. ZFN binding site is underlined. BglII sites are highlighted in blue. Indels are highlighted in red.
Figure Legend Snippet: Targeted insertion of a restriction site using ZFNc-kit and gene-targeting ssODNs. a. A schematic of the mouse c-kit locus and the ssODN sequences of different sizes designed to introduce an exogenous BglII restriction site (in blue) into the genome in vivo using ZFNc-kit and ssODNs. b. Detection of introduced BglII site in founder animals. The representative gel electropherogram of genotyping results by BglII digestion of PCR product. F0 pups were genotyped by BglII digestion of the 592 bp PCR product amplified from tail genomic DNA. The PCR product from the wild type c-kit allele cannot be digested by BglII (WT), while the PCR product from the targeted mutant allele (10 out of 109 founders) can be digested by BglII into two distinct fragments (238 bp and 354 bp). The PCR primers are listed in Table S1 . c. Sequence analysis of the c-kit mutant allele of the 10 founders with correct BglII restriction bands. The target region of the mouse c-kit gene was PCR-amplified from tail genomic DNA of the 5-day-old founders and subjected to T-A cloning. Colonies were randomly picked for DNA sequencing. Precise BglII site insertion was detected in 8 out of 10 mutant founders. ZFN binding site is underlined. BglII sites are highlighted in blue. Indels are highlighted in red.

Techniques Used: Introduce, In Vivo, Polymerase Chain Reaction, Amplification, Mutagenesis, Sequencing, Clone Assay, DNA Sequencing, Binding Assay

Germline transmission of the targeting modification. a. The representative gel electropherogram of the genotyping result by BglII digestion of the PCR product. F1 mice derived from founders C4, #7-1, and A22 were genotyped by BglII digestion of the PCR product (592 bp) amplified from tail genomic DNA of 5-day-old pups. The PCR product from the wild type c-kit allele cannot be digested by BglII (WT), while the targeted mutant allele can be digested by BglII into two distinct fragments (238 bp and 354 bp). The PCR primers are listed in Table S1 . b. Sequence analysis of the BglII restriction site insertion. The F1 mice 1 2 from founder C4, #1 2 from founder 7-1, and #3 8 from founder A22 were selected for sequence analysis to confirm precise restrict site insertion. ZFN binding site is underlined. BglII sites are highlighted in blue. WT, wild type. c. The representative gel electropherogram of the genotyping results by PCR amplification. A total of 13 F1 mice from founder 57 59 with loxP integration were genotyped by PCR amplification using tail genomic DNA from 5-day-old pups. The expected wild type (WT) band is 178 bp, while the expected mutant band is 214 bp. The PCR primers are listed in Table S1 . Ng, negative control. d. Sequence analysis of the mutant c-kit allele of the F1 mice 1, 3, 7, and 8 from founder 57, and #3 4 from founder 59. The targeted region of the mouse c-kit locus was PCR-amplified from tail genomic DNA of 5-day-old founders and subjected to sequencing. Precise loxP integration was detected in F1 mice 1 3 from founder 57, 3 4 from founder 59. ZFN binding site is underlined. loxP sites are highlighted in blue. Deletions are highlighted in red. WT, wild type.
Figure Legend Snippet: Germline transmission of the targeting modification. a. The representative gel electropherogram of the genotyping result by BglII digestion of the PCR product. F1 mice derived from founders C4, #7-1, and A22 were genotyped by BglII digestion of the PCR product (592 bp) amplified from tail genomic DNA of 5-day-old pups. The PCR product from the wild type c-kit allele cannot be digested by BglII (WT), while the targeted mutant allele can be digested by BglII into two distinct fragments (238 bp and 354 bp). The PCR primers are listed in Table S1 . b. Sequence analysis of the BglII restriction site insertion. The F1 mice 1 2 from founder C4, #1 2 from founder 7-1, and #3 8 from founder A22 were selected for sequence analysis to confirm precise restrict site insertion. ZFN binding site is underlined. BglII sites are highlighted in blue. WT, wild type. c. The representative gel electropherogram of the genotyping results by PCR amplification. A total of 13 F1 mice from founder 57 59 with loxP integration were genotyped by PCR amplification using tail genomic DNA from 5-day-old pups. The expected wild type (WT) band is 178 bp, while the expected mutant band is 214 bp. The PCR primers are listed in Table S1 . Ng, negative control. d. Sequence analysis of the mutant c-kit allele of the F1 mice 1, 3, 7, and 8 from founder 57, and #3 4 from founder 59. The targeted region of the mouse c-kit locus was PCR-amplified from tail genomic DNA of 5-day-old founders and subjected to sequencing. Precise loxP integration was detected in F1 mice 1 3 from founder 57, 3 4 from founder 59. ZFN binding site is underlined. loxP sites are highlighted in blue. Deletions are highlighted in red. WT, wild type.

Techniques Used: Transmission Assay, Modification, Polymerase Chain Reaction, Mouse Assay, Derivative Assay, Amplification, Mutagenesis, Sequencing, Binding Assay, Negative Control

5) Product Images from "Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep"

Article Title: Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep

Journal: Scientific Reports

doi: 10.1038/srep32271

Evaluation of sgRNA:Cas9-mediated genetic modifications in lambs. ( a ) PCR products of the targeted region of the MSTN , ASIP , and BCO2 genes of founder sheep co-microinjected with a mixture of Cas9 mRNA and sgRNAs. D2000 DNA Marker was used as a marker reference. ( b ) Detection of sgRNA:Cas9-mediated on-target cleavage of the MSTN , ASIP and BCO2 genes by using a T7EI cleavage assay. All PCR products from ( a ) were subjected to the T7EI cleavage assay. ( c ) Sequencing results of the modified MSTN , ASIP and BCO2 loci that were detected in the founder animals. Target sequences complementary to sgRNAs of targeted genes are in red text, while the PAM sequences are marked in green. The mutations are marked in blue, dashlines indicates deletions, and lowercase indicates insertions or replacements. Insertions (+), deletions (−), mutations (m) is shown to the right of each allele. The genotypes are shown to the right with the rates of total clones for TA-sequencing.
Figure Legend Snippet: Evaluation of sgRNA:Cas9-mediated genetic modifications in lambs. ( a ) PCR products of the targeted region of the MSTN , ASIP , and BCO2 genes of founder sheep co-microinjected with a mixture of Cas9 mRNA and sgRNAs. D2000 DNA Marker was used as a marker reference. ( b ) Detection of sgRNA:Cas9-mediated on-target cleavage of the MSTN , ASIP and BCO2 genes by using a T7EI cleavage assay. All PCR products from ( a ) were subjected to the T7EI cleavage assay. ( c ) Sequencing results of the modified MSTN , ASIP and BCO2 loci that were detected in the founder animals. Target sequences complementary to sgRNAs of targeted genes are in red text, while the PAM sequences are marked in green. The mutations are marked in blue, dashlines indicates deletions, and lowercase indicates insertions or replacements. Insertions (+), deletions (−), mutations (m) is shown to the right of each allele. The genotypes are shown to the right with the rates of total clones for TA-sequencing.

Techniques Used: Polymerase Chain Reaction, Marker, Cleavage Assay, Sequencing, Modification, Clone Assay

6) Product Images from "Distribution of Endophytic Bacteria in Alopecurus aequalis Sobol and Oxalis corniculata L. from Soils Contaminated by Polycyclic Aromatic Hydrocarbons"

Article Title: Distribution of Endophytic Bacteria in Alopecurus aequalis Sobol and Oxalis corniculata L. from Soils Contaminated by Polycyclic Aromatic Hydrocarbons

Journal: PLoS ONE

doi: 10.1371/journal.pone.0083054

Representative DGGE for PCR-amplified 16S rDNA V3 fragments from endophytic bacteria in Alopecurus aequalis and Oxalis corniculata . The genus of the band marked in the figure: Band 1 - Pseudomonas sp; Band 2 - uncultured bacterium clone; Band 3 - Nesterenkonia sp.
Figure Legend Snippet: Representative DGGE for PCR-amplified 16S rDNA V3 fragments from endophytic bacteria in Alopecurus aequalis and Oxalis corniculata . The genus of the band marked in the figure: Band 1 - Pseudomonas sp; Band 2 - uncultured bacterium clone; Band 3 - Nesterenkonia sp.

Techniques Used: Denaturing Gradient Gel Electrophoresis, Polymerase Chain Reaction, Amplification

7) Product Images from "Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system"

Article Title: Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system

Journal: Scientific Reports

doi: 10.1038/srep13878

Detection of the MSTN and FGF5 sgRNA:Cas9-mediated off-target cleavages in vivo. ( a ) PCR products of the potential off-target sites of MSTN and FGF5 sgRNA:Cas9 from founder lambs. A total of 13 potential off-target sites most homologous to MSTN and FGF5 sgRNA were named OT1 to OT13. OT5 and OT7 were selected and PCR amplified from genomic DNA from founders. ( b ) Detection of sgRNA:Cas9-mediated off-target cleavage of MSTN and FGF5 by T7E1 cleavage assay. All PCR products from ( a ) were subjected to T7E1 cleavage assay. ( c ) Sequencing results of PCR products.
Figure Legend Snippet: Detection of the MSTN and FGF5 sgRNA:Cas9-mediated off-target cleavages in vivo. ( a ) PCR products of the potential off-target sites of MSTN and FGF5 sgRNA:Cas9 from founder lambs. A total of 13 potential off-target sites most homologous to MSTN and FGF5 sgRNA were named OT1 to OT13. OT5 and OT7 were selected and PCR amplified from genomic DNA from founders. ( b ) Detection of sgRNA:Cas9-mediated off-target cleavage of MSTN and FGF5 by T7E1 cleavage assay. All PCR products from ( a ) were subjected to T7E1 cleavage assay. ( c ) Sequencing results of PCR products.

Techniques Used: In Vivo, Polymerase Chain Reaction, Amplification, Cleavage Assay, Sequencing

Related Articles

Amplification:

Article Title: Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system
Article Snippet: .. Briefly, the targeted fragments were amplified by PrimerSTAR HS DNA polymerase (TaKaRa, DR010A) from the genomic DNA, then purified with a PCR cleanup kit (Axygen, AP-PCR-50). ..

Article Title: Efficient Knockin Mouse Generation by ssDNA Oligonucleotides and Zinc-Finger Nuclease Assisted Homologous Recombination in Zygotes
Article Snippet: .. In brief, targeted fragments were amplified from extracted DNA, and purified using a PCR cleanup kit (Axygen, AP-PCR-50). .. Purified PCR products were denatured and re-annealed in NEB buffer 2 (NEB) using a thermocycler.

Article Title: Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep
Article Snippet: .. Briefly, the targeted fragments were amplified using PrimerSTAR HS DNA polymerase (TaKaRa, DR010A), then purified with a PCR cleanup kit (Axygen, AP-PCR-50). ..

Article Title: Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep
Article Snippet: .. Briefly, targeted fragments were amplified using PrimerSTAR HS DNA polymerase (TaKaRa, DR010A), then purified with a PCR cleanup kit (Axygen, AP-PCR-50). .. The purified PCR products were denatured and re-annealed in NEBuffer 2 (NEB) using a BioRad thermocycler.

In Vitro:

Article Title: Identification and Functional Analysis of the Novel ORF4 Protein Encoded by Porcine Circovirus Type 2
Article Snippet: .. To synthesize the ORF4 RNA probes, linearized pCI-neo-ORF4 digested with SalI or EcoRI was purified with the PCR cleanup kit (Axygen Biosciences) and then used as the template for synthesis of DIG-labeled ORF4-specific RNA sequence probes by in vitro transcription with T7 or T3 RNA polymerase and an RNA-DIG labeling mix (Roche) according to the manufacturer's protocol. .. The synthesized RNA probes were designated probes S1 and S2.

Labeling:

Article Title: Identification and Functional Analysis of the Novel ORF4 Protein Encoded by Porcine Circovirus Type 2
Article Snippet: .. To synthesize the ORF4 RNA probes, linearized pCI-neo-ORF4 digested with SalI or EcoRI was purified with the PCR cleanup kit (Axygen Biosciences) and then used as the template for synthesis of DIG-labeled ORF4-specific RNA sequence probes by in vitro transcription with T7 or T3 RNA polymerase and an RNA-DIG labeling mix (Roche) according to the manufacturer's protocol. .. The synthesized RNA probes were designated probes S1 and S2.

Purification:

Article Title: Distribution of Endophytic Bacteria in Alopecurus aequalis Sobol and Oxalis corniculata L. from Soils Contaminated by Polycyclic Aromatic Hydrocarbons
Article Snippet: .. If a single band appeared in a DGGE gel for one sample, the PCR products were purified with the PCR Cleanup Kit (Axygen, USA) and used for direct sequencing (Invitrogen). .. When multiple bands appeared in one sample, the bands were repeatedly electrophoresed and excised until only a single band was detectable on the DGGE gel.

Article Title: Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system
Article Snippet: .. Briefly, the targeted fragments were amplified by PrimerSTAR HS DNA polymerase (TaKaRa, DR010A) from the genomic DNA, then purified with a PCR cleanup kit (Axygen, AP-PCR-50). ..

Article Title: Identification and Functional Analysis of the Novel ORF4 Protein Encoded by Porcine Circovirus Type 2
Article Snippet: .. To synthesize the ORF4 RNA probes, linearized pCI-neo-ORF4 digested with SalI or EcoRI was purified with the PCR cleanup kit (Axygen Biosciences) and then used as the template for synthesis of DIG-labeled ORF4-specific RNA sequence probes by in vitro transcription with T7 or T3 RNA polymerase and an RNA-DIG labeling mix (Roche) according to the manufacturer's protocol. .. The synthesized RNA probes were designated probes S1 and S2.

Article Title: CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients
Article Snippet: .. The T7EN cleavage assay was performed as follows: briefly, targeted regions of PD1 were PCR-amplified from genomic DNA using rTaq (Takara, DR001BM) and the products were purified with a PCR cleanup kit (Axygen, APPCR-50). .. Purified PCR product was denatured and re-annealed in NEBuffer 2 (NEB) using a thermocycler (ABI, Veriti9902).

Article Title: Efficient Knockin Mouse Generation by ssDNA Oligonucleotides and Zinc-Finger Nuclease Assisted Homologous Recombination in Zygotes
Article Snippet: .. In brief, targeted fragments were amplified from extracted DNA, and purified using a PCR cleanup kit (Axygen, AP-PCR-50). .. Purified PCR products were denatured and re-annealed in NEB buffer 2 (NEB) using a thermocycler.

Article Title: Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep
Article Snippet: .. Briefly, the targeted fragments were amplified using PrimerSTAR HS DNA polymerase (TaKaRa, DR010A), then purified with a PCR cleanup kit (Axygen, AP-PCR-50). ..

Article Title: Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep
Article Snippet: .. Briefly, targeted fragments were amplified using PrimerSTAR HS DNA polymerase (TaKaRa, DR010A), then purified with a PCR cleanup kit (Axygen, AP-PCR-50). .. The purified PCR products were denatured and re-annealed in NEBuffer 2 (NEB) using a BioRad thermocycler.

Article Title: Involvement of PACLOBUTRAZOL RESISTANCE6/KIDARI, an Atypical bHLH Transcription Factor, in Auxin Responses in Arabidopsis
Article Snippet: .. After washing, the DNA-protein cross-links obtained were reversed at 65°C for 12 h, and DNA was purified using PCR Cleanup Kit (Axygen) for PCR reactions. .. Microscopy Photographs of the seedlings and the GUS stained tissues and organs were taken under a Motic K microscope equipped with an EOS 1100D digital camera.

Polymerase Chain Reaction:

Article Title: Distribution of Endophytic Bacteria in Alopecurus aequalis Sobol and Oxalis corniculata L. from Soils Contaminated by Polycyclic Aromatic Hydrocarbons
Article Snippet: .. If a single band appeared in a DGGE gel for one sample, the PCR products were purified with the PCR Cleanup Kit (Axygen, USA) and used for direct sequencing (Invitrogen). .. When multiple bands appeared in one sample, the bands were repeatedly electrophoresed and excised until only a single band was detectable on the DGGE gel.

Article Title: Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system
Article Snippet: .. Briefly, the targeted fragments were amplified by PrimerSTAR HS DNA polymerase (TaKaRa, DR010A) from the genomic DNA, then purified with a PCR cleanup kit (Axygen, AP-PCR-50). ..

Article Title: Identification and Functional Analysis of the Novel ORF4 Protein Encoded by Porcine Circovirus Type 2
Article Snippet: .. To synthesize the ORF4 RNA probes, linearized pCI-neo-ORF4 digested with SalI or EcoRI was purified with the PCR cleanup kit (Axygen Biosciences) and then used as the template for synthesis of DIG-labeled ORF4-specific RNA sequence probes by in vitro transcription with T7 or T3 RNA polymerase and an RNA-DIG labeling mix (Roche) according to the manufacturer's protocol. .. The synthesized RNA probes were designated probes S1 and S2.

Article Title: CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients
Article Snippet: .. The T7EN cleavage assay was performed as follows: briefly, targeted regions of PD1 were PCR-amplified from genomic DNA using rTaq (Takara, DR001BM) and the products were purified with a PCR cleanup kit (Axygen, APPCR-50). .. Purified PCR product was denatured and re-annealed in NEBuffer 2 (NEB) using a thermocycler (ABI, Veriti9902).

Article Title: Efficient Knockin Mouse Generation by ssDNA Oligonucleotides and Zinc-Finger Nuclease Assisted Homologous Recombination in Zygotes
Article Snippet: .. In brief, targeted fragments were amplified from extracted DNA, and purified using a PCR cleanup kit (Axygen, AP-PCR-50). .. Purified PCR products were denatured and re-annealed in NEB buffer 2 (NEB) using a thermocycler.

Article Title: Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep
Article Snippet: .. Briefly, the targeted fragments were amplified using PrimerSTAR HS DNA polymerase (TaKaRa, DR010A), then purified with a PCR cleanup kit (Axygen, AP-PCR-50). ..

Article Title: Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep
Article Snippet: .. Briefly, targeted fragments were amplified using PrimerSTAR HS DNA polymerase (TaKaRa, DR010A), then purified with a PCR cleanup kit (Axygen, AP-PCR-50). .. The purified PCR products were denatured and re-annealed in NEBuffer 2 (NEB) using a BioRad thermocycler.

Article Title: Involvement of PACLOBUTRAZOL RESISTANCE6/KIDARI, an Atypical bHLH Transcription Factor, in Auxin Responses in Arabidopsis
Article Snippet: .. After washing, the DNA-protein cross-links obtained were reversed at 65°C for 12 h, and DNA was purified using PCR Cleanup Kit (Axygen) for PCR reactions. .. Microscopy Photographs of the seedlings and the GUS stained tissues and organs were taken under a Motic K microscope equipped with an EOS 1100D digital camera.

Sequencing:

Article Title: Distribution of Endophytic Bacteria in Alopecurus aequalis Sobol and Oxalis corniculata L. from Soils Contaminated by Polycyclic Aromatic Hydrocarbons
Article Snippet: .. If a single band appeared in a DGGE gel for one sample, the PCR products were purified with the PCR Cleanup Kit (Axygen, USA) and used for direct sequencing (Invitrogen). .. When multiple bands appeared in one sample, the bands were repeatedly electrophoresed and excised until only a single band was detectable on the DGGE gel.

Article Title: Identification and Functional Analysis of the Novel ORF4 Protein Encoded by Porcine Circovirus Type 2
Article Snippet: .. To synthesize the ORF4 RNA probes, linearized pCI-neo-ORF4 digested with SalI or EcoRI was purified with the PCR cleanup kit (Axygen Biosciences) and then used as the template for synthesis of DIG-labeled ORF4-specific RNA sequence probes by in vitro transcription with T7 or T3 RNA polymerase and an RNA-DIG labeling mix (Roche) according to the manufacturer's protocol. .. The synthesized RNA probes were designated probes S1 and S2.

Denaturing Gradient Gel Electrophoresis:

Article Title: Distribution of Endophytic Bacteria in Alopecurus aequalis Sobol and Oxalis corniculata L. from Soils Contaminated by Polycyclic Aromatic Hydrocarbons
Article Snippet: .. If a single band appeared in a DGGE gel for one sample, the PCR products were purified with the PCR Cleanup Kit (Axygen, USA) and used for direct sequencing (Invitrogen). .. When multiple bands appeared in one sample, the bands were repeatedly electrophoresed and excised until only a single band was detectable on the DGGE gel.

Cleavage Assay:

Article Title: CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients
Article Snippet: .. The T7EN cleavage assay was performed as follows: briefly, targeted regions of PD1 were PCR-amplified from genomic DNA using rTaq (Takara, DR001BM) and the products were purified with a PCR cleanup kit (Axygen, APPCR-50). .. Purified PCR product was denatured and re-annealed in NEBuffer 2 (NEB) using a thermocycler (ABI, Veriti9902).

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    Axygen pcr cleanup kit
    Phenotypes of the pre6 mutants and the PRE6 overexpression plants. (A) A diagram showing the <t>T-DNA</t> insertion sites in the pre6-1 and pre6-2 mutants. (B) Expression of PRE6 transcript in the pre6 mutants ( pre6-1 and pre6-2 ) and transgenic plants overexpressing PRE6 ( 35S:PRE6-1 and 35S:PRE6-2 ). RNA was isolated from 14-day-old seedlings, and <t>RT-PCR</t> was used to examine the expression of PRE6 . The expression of ACT2 was used as a control. (C) Photographs of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Sterilized seeds were grown on vertically placed 1/2 MS plates in darkness, and photographs were taken under a microscope. (D) Hypocotyl length of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Photographs were taken under a microscope, and ImageJ software was used to measure the hypocotyl length. Data represent the mean ± SD of 33–35 seedlings. ∗ Significantly different from the Col wild type seedlings ( p
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    Phenotypes of the pre6 mutants and the PRE6 overexpression plants. (A) A diagram showing the T-DNA insertion sites in the pre6-1 and pre6-2 mutants. (B) Expression of PRE6 transcript in the pre6 mutants ( pre6-1 and pre6-2 ) and transgenic plants overexpressing PRE6 ( 35S:PRE6-1 and 35S:PRE6-2 ). RNA was isolated from 14-day-old seedlings, and RT-PCR was used to examine the expression of PRE6 . The expression of ACT2 was used as a control. (C) Photographs of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Sterilized seeds were grown on vertically placed 1/2 MS plates in darkness, and photographs were taken under a microscope. (D) Hypocotyl length of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Photographs were taken under a microscope, and ImageJ software was used to measure the hypocotyl length. Data represent the mean ± SD of 33–35 seedlings. ∗ Significantly different from the Col wild type seedlings ( p

    Journal: Frontiers in Plant Science

    Article Title: Involvement of PACLOBUTRAZOL RESISTANCE6/KIDARI, an Atypical bHLH Transcription Factor, in Auxin Responses in Arabidopsis

    doi: 10.3389/fpls.2017.01813

    Figure Lengend Snippet: Phenotypes of the pre6 mutants and the PRE6 overexpression plants. (A) A diagram showing the T-DNA insertion sites in the pre6-1 and pre6-2 mutants. (B) Expression of PRE6 transcript in the pre6 mutants ( pre6-1 and pre6-2 ) and transgenic plants overexpressing PRE6 ( 35S:PRE6-1 and 35S:PRE6-2 ). RNA was isolated from 14-day-old seedlings, and RT-PCR was used to examine the expression of PRE6 . The expression of ACT2 was used as a control. (C) Photographs of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Sterilized seeds were grown on vertically placed 1/2 MS plates in darkness, and photographs were taken under a microscope. (D) Hypocotyl length of the 5-day-old etiolated seedlings of the Col wild type, the pre6 mutants and the 35S:PRE6 transgenic plants. Photographs were taken under a microscope, and ImageJ software was used to measure the hypocotyl length. Data represent the mean ± SD of 33–35 seedlings. ∗ Significantly different from the Col wild type seedlings ( p

    Article Snippet: After washing, the DNA-protein cross-links obtained were reversed at 65°C for 12 h, and DNA was purified using PCR Cleanup Kit (Axygen) for PCR reactions.

    Techniques: Over Expression, Expressing, Transgenic Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Mass Spectrometry, Microscopy, Software

    Evaluation of sgRNA:Cas9-mediated genetic modifications in lambs. ( a ) PCR products of the targeted region of the MSTN , ASIP , and BCO2 genes of founder sheep co-microinjected with a mixture of Cas9 mRNA and sgRNAs. D2000 DNA Marker was used as a marker reference. ( b ) Detection of sgRNA:Cas9-mediated on-target cleavage of the MSTN , ASIP and BCO2 genes by using a T7EI cleavage assay. All PCR products from ( a ) were subjected to the T7EI cleavage assay. ( c ) Sequencing results of the modified MSTN , ASIP and BCO2 loci that were detected in the founder animals. Target sequences complementary to sgRNAs of targeted genes are in red text, while the PAM sequences are marked in green. The mutations are marked in blue, dashlines indicates deletions, and lowercase indicates insertions or replacements. Insertions (+), deletions (−), mutations (m) is shown to the right of each allele. The genotypes are shown to the right with the rates of total clones for TA-sequencing.

    Journal: Scientific Reports

    Article Title: Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep

    doi: 10.1038/srep32271

    Figure Lengend Snippet: Evaluation of sgRNA:Cas9-mediated genetic modifications in lambs. ( a ) PCR products of the targeted region of the MSTN , ASIP , and BCO2 genes of founder sheep co-microinjected with a mixture of Cas9 mRNA and sgRNAs. D2000 DNA Marker was used as a marker reference. ( b ) Detection of sgRNA:Cas9-mediated on-target cleavage of the MSTN , ASIP and BCO2 genes by using a T7EI cleavage assay. All PCR products from ( a ) were subjected to the T7EI cleavage assay. ( c ) Sequencing results of the modified MSTN , ASIP and BCO2 loci that were detected in the founder animals. Target sequences complementary to sgRNAs of targeted genes are in red text, while the PAM sequences are marked in green. The mutations are marked in blue, dashlines indicates deletions, and lowercase indicates insertions or replacements. Insertions (+), deletions (−), mutations (m) is shown to the right of each allele. The genotypes are shown to the right with the rates of total clones for TA-sequencing.

    Article Snippet: Briefly, the targeted fragments were amplified using PrimerSTAR HS DNA polymerase (TaKaRa, DR010A), then purified with a PCR cleanup kit (Axygen, AP-PCR-50).

    Techniques: Polymerase Chain Reaction, Marker, Cleavage Assay, Sequencing, Modification, Clone Assay

    Cas9 mediated efficient hPD-1 KO in primary human T cells of healthy donors and patients. Freshly isolated PBMC were activated in vitro by IFN-γ for 3 d and IL-2 and aCD3 for 2 d and were transfected with pST1374-Cas9-GFP and pGL3-U6-hPD-1-sgRNA plasmids for each reaction. Sample G and Z stand for two individual patients and H stands for a healthy donor. ( a ) The GFP expression was evaluated by fluorescence microscope 24 h after electroporation (Donor 1 and Donor 2 are patients. Donor 3 is a representative of healthy donor). ( b ) PCR products were amplified and subjected to T7EN1 cleavage assay. Samples with cleavage bands were marked with an asterisk “*”. Sample G1/H1/Z1 represent control T cells and G2/H2/Z2 represent hPD-1 KO T cells. “ + ” represents for the positive control with the cleavage bands detected on HeLa cell line. NC, negative control. ( c ) DNA sequences of marked samples. TA clones from the PCR products were analyzed by DNA sequencing. The PAM sequences are underlined and highlighted in green; the targeting sequences in red; the mutations in blue, lower case; deletions (−), and insertions (+). The above experiments have been repeated 3 times with similar results.

    Journal: Scientific Reports

    Article Title: CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients

    doi: 10.1038/srep20070

    Figure Lengend Snippet: Cas9 mediated efficient hPD-1 KO in primary human T cells of healthy donors and patients. Freshly isolated PBMC were activated in vitro by IFN-γ for 3 d and IL-2 and aCD3 for 2 d and were transfected with pST1374-Cas9-GFP and pGL3-U6-hPD-1-sgRNA plasmids for each reaction. Sample G and Z stand for two individual patients and H stands for a healthy donor. ( a ) The GFP expression was evaluated by fluorescence microscope 24 h after electroporation (Donor 1 and Donor 2 are patients. Donor 3 is a representative of healthy donor). ( b ) PCR products were amplified and subjected to T7EN1 cleavage assay. Samples with cleavage bands were marked with an asterisk “*”. Sample G1/H1/Z1 represent control T cells and G2/H2/Z2 represent hPD-1 KO T cells. “ + ” represents for the positive control with the cleavage bands detected on HeLa cell line. NC, negative control. ( c ) DNA sequences of marked samples. TA clones from the PCR products were analyzed by DNA sequencing. The PAM sequences are underlined and highlighted in green; the targeting sequences in red; the mutations in blue, lower case; deletions (−), and insertions (+). The above experiments have been repeated 3 times with similar results.

    Article Snippet: The T7EN cleavage assay was performed as follows: briefly, targeted regions of PD1 were PCR-amplified from genomic DNA using rTaq (Takara, DR001BM) and the products were purified with a PCR cleanup kit (Axygen, APPCR-50).

    Techniques: Isolation, In Vitro, Transfection, Expressing, Fluorescence, Microscopy, Electroporation, Polymerase Chain Reaction, Amplification, Cleavage Assay, Positive Control, Negative Control, Clone Assay, DNA Sequencing

    Evaluation of hPD-1 sgRNA:Cas9-mediated modifications of human PD-1. ( a ) Schematic diagram of sgRNAs targeting at hPD-1 Exon 2 locus. The sgRNAs targeting sites on the sense strand are colored with blue while those on the antisense are colored with red. PAM sequences are underlined. ( b ) Detection of sgRNA:Cas9-mediated cleavage of hPD-1 by PCR and T7EN1 cleavage assay. M, DNA marker. sg1, sgRNA 1; sg2, sgRNA 2; sg3, sgRNA 3; sg4, sgRNA 4; sg (1 + 2), sgRNA 1 combined with sgRNA 2; sg (3 + 4), sgRNA 3 combined with sgRNA 4. Con, negative control. The above experiments have been repeated 3 times with similar results.

    Journal: Scientific Reports

    Article Title: CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients

    doi: 10.1038/srep20070

    Figure Lengend Snippet: Evaluation of hPD-1 sgRNA:Cas9-mediated modifications of human PD-1. ( a ) Schematic diagram of sgRNAs targeting at hPD-1 Exon 2 locus. The sgRNAs targeting sites on the sense strand are colored with blue while those on the antisense are colored with red. PAM sequences are underlined. ( b ) Detection of sgRNA:Cas9-mediated cleavage of hPD-1 by PCR and T7EN1 cleavage assay. M, DNA marker. sg1, sgRNA 1; sg2, sgRNA 2; sg3, sgRNA 3; sg4, sgRNA 4; sg (1 + 2), sgRNA 1 combined with sgRNA 2; sg (3 + 4), sgRNA 3 combined with sgRNA 4. Con, negative control. The above experiments have been repeated 3 times with similar results.

    Article Snippet: The T7EN cleavage assay was performed as follows: briefly, targeted regions of PD1 were PCR-amplified from genomic DNA using rTaq (Takara, DR001BM) and the products were purified with a PCR cleanup kit (Axygen, APPCR-50).

    Techniques: Polymerase Chain Reaction, Cleavage Assay, Marker, Negative Control

    Generation of ZFN-mediated c-kit mutant mice. a. Schematic showing the layout of ZFN target sites. The left and right ZFN binding sites with the spacer region are shown. b. Sequence analysis of the modified c-kit allele in 44 founder animals. The targeted region on the mouse c-kit locus was PCR-amplified from genomic DNA extracted from tails of the 5-day-old F0 pups, then subjected to T-A cloning. Colonies were randomly picked for DNA sequencing. Indels and mutations were detected in 8 out of 27 founders treated with 2.5 ng/μl ZFN mRNA and 7 out of 17 founders treated with 5 ng/μl ZFN mRNA. The ZFN binding site is underlined. Indels and mutations are highlighted in red.

    Journal: PLoS ONE

    Article Title: Efficient Knockin Mouse Generation by ssDNA Oligonucleotides and Zinc-Finger Nuclease Assisted Homologous Recombination in Zygotes

    doi: 10.1371/journal.pone.0077696

    Figure Lengend Snippet: Generation of ZFN-mediated c-kit mutant mice. a. Schematic showing the layout of ZFN target sites. The left and right ZFN binding sites with the spacer region are shown. b. Sequence analysis of the modified c-kit allele in 44 founder animals. The targeted region on the mouse c-kit locus was PCR-amplified from genomic DNA extracted from tails of the 5-day-old F0 pups, then subjected to T-A cloning. Colonies were randomly picked for DNA sequencing. Indels and mutations were detected in 8 out of 27 founders treated with 2.5 ng/μl ZFN mRNA and 7 out of 17 founders treated with 5 ng/μl ZFN mRNA. The ZFN binding site is underlined. Indels and mutations are highlighted in red.

    Article Snippet: In brief, targeted fragments were amplified from extracted DNA, and purified using a PCR cleanup kit (Axygen, AP-PCR-50).

    Techniques: Mutagenesis, Mouse Assay, Binding Assay, Sequencing, Modification, Polymerase Chain Reaction, Amplification, Clone Assay, DNA Sequencing

    Targeted integration of a loxP site using ZFNc-kit and gene-targeting ssODNs . a. A schematic of the mouse c-kit locus and the ssODN sequences used to introduce a loxP site (in blue) into the genome in vivo . b. The representative gel electropherogram of the genotyping results by PCR amplification. 5 day old pups were genotyped by PCR amplification of tail genomic DNA. The expected wild type band is 178 bp (WT) and the expected mutant band is 214 bp. The PCR primers are listed in Table S1 . Ng, negative control. c. Sequence analysis of the mutant c-kit allele of the 9 founders. The target region of the mouse c-kit gene was PCR-amplified from tail genomic DNA of the 5-day-old founders and subjected to T-A cloning. Colonies were randomly picked for DNA sequencing. Precise loxP site insertion was detected in 2 out of 9 founders. ZFN binding site is underlined. The loxP sites are highlighted in blue. Indels are highlighted in red. WT, wild type.

    Journal: PLoS ONE

    Article Title: Efficient Knockin Mouse Generation by ssDNA Oligonucleotides and Zinc-Finger Nuclease Assisted Homologous Recombination in Zygotes

    doi: 10.1371/journal.pone.0077696

    Figure Lengend Snippet: Targeted integration of a loxP site using ZFNc-kit and gene-targeting ssODNs . a. A schematic of the mouse c-kit locus and the ssODN sequences used to introduce a loxP site (in blue) into the genome in vivo . b. The representative gel electropherogram of the genotyping results by PCR amplification. 5 day old pups were genotyped by PCR amplification of tail genomic DNA. The expected wild type band is 178 bp (WT) and the expected mutant band is 214 bp. The PCR primers are listed in Table S1 . Ng, negative control. c. Sequence analysis of the mutant c-kit allele of the 9 founders. The target region of the mouse c-kit gene was PCR-amplified from tail genomic DNA of the 5-day-old founders and subjected to T-A cloning. Colonies were randomly picked for DNA sequencing. Precise loxP site insertion was detected in 2 out of 9 founders. ZFN binding site is underlined. The loxP sites are highlighted in blue. Indels are highlighted in red. WT, wild type.

    Article Snippet: In brief, targeted fragments were amplified from extracted DNA, and purified using a PCR cleanup kit (Axygen, AP-PCR-50).

    Techniques: Introduce, In Vivo, Polymerase Chain Reaction, Amplification, Mutagenesis, Negative Control, Sequencing, Clone Assay, DNA Sequencing, Binding Assay

    Targeted insertion of a restriction site using ZFNc-kit and gene-targeting ssODNs. a. A schematic of the mouse c-kit locus and the ssODN sequences of different sizes designed to introduce an exogenous BglII restriction site (in blue) into the genome in vivo using ZFNc-kit and ssODNs. b. Detection of introduced BglII site in founder animals. The representative gel electropherogram of genotyping results by BglII digestion of PCR product. F0 pups were genotyped by BglII digestion of the 592 bp PCR product amplified from tail genomic DNA. The PCR product from the wild type c-kit allele cannot be digested by BglII (WT), while the PCR product from the targeted mutant allele (10 out of 109 founders) can be digested by BglII into two distinct fragments (238 bp and 354 bp). The PCR primers are listed in Table S1 . c. Sequence analysis of the c-kit mutant allele of the 10 founders with correct BglII restriction bands. The target region of the mouse c-kit gene was PCR-amplified from tail genomic DNA of the 5-day-old founders and subjected to T-A cloning. Colonies were randomly picked for DNA sequencing. Precise BglII site insertion was detected in 8 out of 10 mutant founders. ZFN binding site is underlined. BglII sites are highlighted in blue. Indels are highlighted in red.

    Journal: PLoS ONE

    Article Title: Efficient Knockin Mouse Generation by ssDNA Oligonucleotides and Zinc-Finger Nuclease Assisted Homologous Recombination in Zygotes

    doi: 10.1371/journal.pone.0077696

    Figure Lengend Snippet: Targeted insertion of a restriction site using ZFNc-kit and gene-targeting ssODNs. a. A schematic of the mouse c-kit locus and the ssODN sequences of different sizes designed to introduce an exogenous BglII restriction site (in blue) into the genome in vivo using ZFNc-kit and ssODNs. b. Detection of introduced BglII site in founder animals. The representative gel electropherogram of genotyping results by BglII digestion of PCR product. F0 pups were genotyped by BglII digestion of the 592 bp PCR product amplified from tail genomic DNA. The PCR product from the wild type c-kit allele cannot be digested by BglII (WT), while the PCR product from the targeted mutant allele (10 out of 109 founders) can be digested by BglII into two distinct fragments (238 bp and 354 bp). The PCR primers are listed in Table S1 . c. Sequence analysis of the c-kit mutant allele of the 10 founders with correct BglII restriction bands. The target region of the mouse c-kit gene was PCR-amplified from tail genomic DNA of the 5-day-old founders and subjected to T-A cloning. Colonies were randomly picked for DNA sequencing. Precise BglII site insertion was detected in 8 out of 10 mutant founders. ZFN binding site is underlined. BglII sites are highlighted in blue. Indels are highlighted in red.

    Article Snippet: In brief, targeted fragments were amplified from extracted DNA, and purified using a PCR cleanup kit (Axygen, AP-PCR-50).

    Techniques: Introduce, In Vivo, Polymerase Chain Reaction, Amplification, Mutagenesis, Sequencing, Clone Assay, DNA Sequencing, Binding Assay

    Germline transmission of the targeting modification. a. The representative gel electropherogram of the genotyping result by BglII digestion of the PCR product. F1 mice derived from founders C4, #7-1, and A22 were genotyped by BglII digestion of the PCR product (592 bp) amplified from tail genomic DNA of 5-day-old pups. The PCR product from the wild type c-kit allele cannot be digested by BglII (WT), while the targeted mutant allele can be digested by BglII into two distinct fragments (238 bp and 354 bp). The PCR primers are listed in Table S1 . b. Sequence analysis of the BglII restriction site insertion. The F1 mice 1 2 from founder C4, #1 2 from founder 7-1, and #3 8 from founder A22 were selected for sequence analysis to confirm precise restrict site insertion. ZFN binding site is underlined. BglII sites are highlighted in blue. WT, wild type. c. The representative gel electropherogram of the genotyping results by PCR amplification. A total of 13 F1 mice from founder 57 59 with loxP integration were genotyped by PCR amplification using tail genomic DNA from 5-day-old pups. The expected wild type (WT) band is 178 bp, while the expected mutant band is 214 bp. The PCR primers are listed in Table S1 . Ng, negative control. d. Sequence analysis of the mutant c-kit allele of the F1 mice 1, 3, 7, and 8 from founder 57, and #3 4 from founder 59. The targeted region of the mouse c-kit locus was PCR-amplified from tail genomic DNA of 5-day-old founders and subjected to sequencing. Precise loxP integration was detected in F1 mice 1 3 from founder 57, 3 4 from founder 59. ZFN binding site is underlined. loxP sites are highlighted in blue. Deletions are highlighted in red. WT, wild type.

    Journal: PLoS ONE

    Article Title: Efficient Knockin Mouse Generation by ssDNA Oligonucleotides and Zinc-Finger Nuclease Assisted Homologous Recombination in Zygotes

    doi: 10.1371/journal.pone.0077696

    Figure Lengend Snippet: Germline transmission of the targeting modification. a. The representative gel electropherogram of the genotyping result by BglII digestion of the PCR product. F1 mice derived from founders C4, #7-1, and A22 were genotyped by BglII digestion of the PCR product (592 bp) amplified from tail genomic DNA of 5-day-old pups. The PCR product from the wild type c-kit allele cannot be digested by BglII (WT), while the targeted mutant allele can be digested by BglII into two distinct fragments (238 bp and 354 bp). The PCR primers are listed in Table S1 . b. Sequence analysis of the BglII restriction site insertion. The F1 mice 1 2 from founder C4, #1 2 from founder 7-1, and #3 8 from founder A22 were selected for sequence analysis to confirm precise restrict site insertion. ZFN binding site is underlined. BglII sites are highlighted in blue. WT, wild type. c. The representative gel electropherogram of the genotyping results by PCR amplification. A total of 13 F1 mice from founder 57 59 with loxP integration were genotyped by PCR amplification using tail genomic DNA from 5-day-old pups. The expected wild type (WT) band is 178 bp, while the expected mutant band is 214 bp. The PCR primers are listed in Table S1 . Ng, negative control. d. Sequence analysis of the mutant c-kit allele of the F1 mice 1, 3, 7, and 8 from founder 57, and #3 4 from founder 59. The targeted region of the mouse c-kit locus was PCR-amplified from tail genomic DNA of 5-day-old founders and subjected to sequencing. Precise loxP integration was detected in F1 mice 1 3 from founder 57, 3 4 from founder 59. ZFN binding site is underlined. loxP sites are highlighted in blue. Deletions are highlighted in red. WT, wild type.

    Article Snippet: In brief, targeted fragments were amplified from extracted DNA, and purified using a PCR cleanup kit (Axygen, AP-PCR-50).

    Techniques: Transmission Assay, Modification, Polymerase Chain Reaction, Mouse Assay, Derivative Assay, Amplification, Mutagenesis, Sequencing, Binding Assay, Negative Control