Hi all,
I have tried to use Crispr Cas9 ( plasmid px333_cas9_mCherry) to delete some enhancers. I designed sgRNA using Chopchop website to target both sides of an enhancer to induce homologous recombination. The deletion fragments are about 1.5 to 2kb. I isolated genomic DNA and did pcr to amplify deletion region, and most of the clones got 2 bands for WT and deletion region, but I selected the 1 band deletion clone with correct band size. Then I did chip, and some of the deletions worked with no signal, but some got signal like WT. I tried the PCR again for genotyping these clones with 2 different primer sets but I only see 1 band. if my cell type is polyploidy would it have complication for Crispr deletion ? Should I make bigger deletion ?
Thank you
Concerning homologous deletion: Deletion may a) be very efficient in cells cotransfected with your plasmid and your sgRNA; b) the other allele may not be properly repaired; c) your cell line may be unstable.
Concerning the deletion, it depends on your goal: If you know enough about the enhancer, your deletions are big enough, however you have to consider that there may be more than on enhancer per gene or additional regulatory elements.
Hi, it can be that your bigger band is not appearing on the pcr because of being bigger (product not favoured). You could design another set of primers to screen for 5' and 3' junction of WT to strongly validate deletion.
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