I am doing crispr knockout in T cells. After transfection, I am trying to validate knockout by western blot and sequencing. with western blot, I can see truncated protein in knockout cells whereas in scrambled cells, band is at the expected position of the target protein.
With agarose gel, I can see 3 bands for knockout protein and scrambled is one band. My question is how do I sequence three bands. Shall i cut individual bands extract and purify DNA and then send for sequncing ?
what should be my approach? I am attaching pic of gel for reference
Hi Richa, Based on my experience I would like to say that no need to cut the band, you can sequence the PCR product. Many times in CRISPR Knock out experiments due to deletion at the target guide RNA binding site following successful Knock out we observe two bands in the gel. As you mentioned you could report a truncated protein, which indicates a knockout event. This is due to biallelic or heterozygous mutations at the target sites.
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