Genomic DNA was extracted using QuickExtract DNA extraction solution (Epicentre) and the target locus amplified by PCR using Phusion High Fidelity DNA Polymerase (Thermo Fisher). Oligos are listed in Supplementary Data 4 . To evaluate indels resulting from cleavage of one gRNA, purified PCR products were sequenced and analyzed using the TIDE or the SYNTHEGO ICE software38 (link),66 . In some experiments DNA editing was measured also by T7 Endonuclease 1 (T7E1) assay (New England BioLabs) following manufacturer’s instructions and as previously described41 (link).
T7 endonuclease 1 t7e1 assay
Manufactured by New England Biolabs
Sourced in United States
T7 Endonuclease 1 (T7E1) is a DNA endonuclease that recognizes and cleaves DNA heteroduplexes. It is useful for detecting single nucleotide polymorphisms (SNPs) and small insertions or deletions (InDels) in DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Quickextract dna extraction solution, by Illumina (1 mentions)
Primestar gxl dna polymerase, by Takara Bio (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Universal dna purification kit, by Tiangen Biotech (1 mentions)
4 protocols using t7 endonuclease 1 t7e1 assay
1
Genomic DNA Extraction and Analysis
2
Optimizing CRISPR-Cas9 Genome Editing
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What Is Known?
• Genome editing is being used increasingly to study gene function.
• CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/ CRISPR-associated protein 9) is a powerful tool that can be used to modify eukaryotic DNA in vivo.
What New Information Does This Article Contribute? Single-stranded sgRNA sequences were annealed and cloned into pSpCas9(BB)-2A-Puro (PX459) after restriction digestion with Bbs1 (PX459 was a gift from Feng Zhang [Addgene plasmid no. 48139]). 21 (link) Successful cloning was confirmed by double digestion with BbsI and AgeI. To determine functionality of sgRNA, sgRNA-PX459 vectors were transfected into NIH-3T3 cells. Genomic DNA was amplified with Taq Roche DNA polymerase (Sigma-Aldrich, Zwijndrecht, The Netherlands) using primers against the modified locus and the top 4 potential gene-coding OT (see Section II in the Online Data Supplement for a list of primers). The presence of indels formation was determined using a T7 endonuclease 1 (T7E1) assay, according to manufacturer's instructions (New England Biolabs, Bioké, Leiden, The Netherlands).
• Genome editing is being used increasingly to study gene function.
• CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/ CRISPR-associated protein 9) is a powerful tool that can be used to modify eukaryotic DNA in vivo.
What New Information Does This Article Contribute? Single-stranded sgRNA sequences were annealed and cloned into pSpCas9(BB)-2A-Puro (PX459) after restriction digestion with Bbs1 (PX459 was a gift from Feng Zhang [Addgene plasmid no. 48139]). 21 (link) Successful cloning was confirmed by double digestion with BbsI and AgeI. To determine functionality of sgRNA, sgRNA-PX459 vectors were transfected into NIH-3T3 cells. Genomic DNA was amplified with Taq Roche DNA polymerase (Sigma-Aldrich, Zwijndrecht, The Netherlands) using primers against the modified locus and the top 4 potential gene-coding OT (see Section II in the Online Data Supplement for a list of primers). The presence of indels formation was determined using a T7 endonuclease 1 (T7E1) assay, according to manufacturer's instructions (New England Biolabs, Bioké, Leiden, The Netherlands).
3
Off-target analysis of CRISPR gRNA
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We designed different primers near the predicted gRNA off‐target sites throughout the whole genome from the online software CCTop. When using CCTop, we chose a custom target selection with in vitro transcription while the species was set as Human (Homo sapiens GRCh37/hg19). Other parameters were unchanged. These fragments of Genomic DNA extracted from iPSCs were amplified with the primers by PrimeSTAR GXL DNA Polymerase (TAKARA) and then purified through Universal DNA Purification Kit (Tiangen). The purified products were used for T7 Endonuclease I (T7E1) assay following the manufacturer's protocol (New England BioLabs) and analysed on 2% agarose gels using Gel Imaging System (Bio‐Rad).
4
Generating mafbb Knockout Mutants
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mafbb knockout mutants were generated through the CRISPR-Cas9 system and performed following the protocol as described in [27 (link)]. Two mafbb specific guided RNAs (as in Figure 1 B) were designed to target the beginning site of the exon. One-cell stage WT embryos were injected with 1 nL of the solution containing 100 ng/µL Cas9 mRNA and 20 ng/µL gRNA. Injected F0 fish were grown to adulthood and outcrossed to WT fish. F1 mutant offsprings were identified with T7 endonuclease I (T7E1) assay (M0302S, NEB, Ipswitch, MA, USA) using primers around the target loci. Each target loci was amplified by PCR from the genomic DNA and the mutation was revealed by DNA sequencing. F1 fish were outcrossed to WT to obtain stable F2 mutant lines. Primers used in T7E1 assay and PCR amplification are listed in Table A1 (Appendix A ).
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