Hi all. I'm trying to create a heterozygous deletion (55bp) mutation in iPSCs to model malignant tranformation. I'm using two sgRNAs in a plasmid based system (puromycin selection). I obtained ~20 clones, extracted genomic DNA, PCR amplified a segment of DNA around the expected deletion and Sanger sequenced the products. My problem was that most clones appear to be homozygous to the mutation. A small minority was negative for any mutation. I confirmed this by reamplifying with a second set for primers and sequncing from both sides with identical results. In other words, the procedure worked to well.
My question is how to obtain the heterozygous clones I need. I know what is recommended for HDR knockin mutation (using mixed donor etc) but I don't know what to do for knockouts. My only idea is using different guides that scored lower for efficiency but I could not find references for that in the literature.
Does anybody know a well tested method?
Thanks for your help
Hello,
I'm having the same issue. I found this paper Article Efficient introduction of specific homozygous and heterozygo...
however, I'm wondering if there is any other way to obtein Htz clones. I would be very interested if you had found an answer to this problem.Thank you
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