Hi everyone, I am trying to use CRISPR/Cas9 to mutate one gene in leukemia cell lines by Lipo3000 (from AGC to AAC). After transfection and puromycin selection of 5 days, the GFP+ of the cells is more than 90%, which means transfection is working. I sent both genomic DNA and cDNA PCR products for sequencing one month after transfection, but the sequencing results showed gDNA has the mutated genotyping (AAC) which is what I want but cDNA is still Wildtype(AGC). It would be highly appreciated If someone could give me some suggestions about the problem.
The vectors I used are HR210PA-1 and CAS940A-1 from SBI.
Thank you very much
Your result with the cDNA suggests that you did not succeed to mutate the targeted gene. With the limited information, I can only guess what happened:
- Random multiple integration of your KI-construct. You can avoid this by using vectors with a PGK-hsvTK selection cassette.
- Contamination of your genomic DNA with the KI-construct.
You may have the chance to design a genomic PCR with primers that are not part of the KI-construct. Then you are safe that you analyze the desired locus.
I think that Uwe Borgmeyer is correct but as an alternative were your no template controls negative?. Pcr contamination of the wild type amplimer might be a problem
To me, I would say your sample is heterozygous, in genomic DNA the sequencer might pick up the mutated allele. In this case, you have to share the chromatogram to see how does it looks like.
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