Genomic DNA Extraction and Genotyping

Genomic DNA was extracted from ear clip biopsies using the DNA Extract All Reagents Kit (Applied Biosystems) according to the manufacturer's instructions. Genotyping primers for SR-PCR assays were chosen to be at least 200 bp away from the sequences targeted by sgRNA, depending on available sequences for design. PCR assays were optimised and performed as previously described (27 (link)). The PCR products were purified employing a QIAquick Gel Extraction Kit (Qiagen) and sent for Sanger sequencing.

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Owens D.D., Caulder A., Frontera V., Harman J.R., Allan A.J., Bucakci A., Greder L., Codner G.F., Hublitz P., McHugh P.J., Teboul L, & de Bruijn M.F. (2019). Microhomologies are prevalent at Cas9-induced larger deletions. Nucleic Acids Research, 47(14), 7402-7417.

Publication 2019

DNA Extract All Reagents Kit QIAquick Gel Extraction Kit

Assays Biopsies Clip Genomic Primers

Corresponding Organization : Mary Lyon Centre at MRC Harwell

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Variable analysis

independent variables

Genomic DNA extraction method (DNA Extract All Reagents Kit)

dependent variables

Genotyping results from SR-PCR assays
Sanger sequencing results

control variables

Primer design (200 bp away from sgRNA target sequences)
PCR assay optimization and performance as previously described

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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