I confused in designing homologous arm for Knock in experiment using crispr cas 9 .
Does people use BAC DNA for human genome sequence and how can i buy BAC DNA for my desired sequence?
Let me bring you some help. I work with CRISPR/Cas9 but in bacteria. Bacteria can't survive with Cas9 and sgRNA at the same time, we need to transform with a donor DNA, so it can recombine at the DSB site.
We amplify by PCR the sequences that flank the gene to be deleted/substituted and then merge into one dsDNA fragment by overlap extension PCR so it's like a knock in.
Hope my answer help.
Thank you Eduardo .So you mean that u isolate the genomic DNA and amplify your homologous arms and then insert it into the Donor DNA
Hi Ahmed. Yes, the best way is to amplify from genomic DNA isolated from whatever you are working with (cells, animals). That way you minimize the possibility of sequence polymorphisms reducing or inhibiting your KI efficiency. GeneCopoeia can help you with this. Visit http://www.genecopoeia.com/product/crispr-cas9/ to learn more.
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