hi,
I have recently knocked out a gene of interest in a mammalian cancer cell line using Santa Cruz Biotechnology knockout and HDR plasmids. I have confirmed the knockout in mRNA and protein levels and submitted my article to a journal. One of referees has asked to confirm the knockout in DNA level too. But considering that the HDR plasmid contains two genes including RFP and puromycin resistance gene (a rather huge segment in total), and that these genes have been inserted to the break site in target sequence in the genome, then the confirmation by sequencing the target site is actually next to impossible. What should I do now or how should I reply to the required revision in my article?
thanks
I would design PCR primers across the junction -- one inside your insertion, and one outside in the genomic area. If they give you the expected length & sequence, and you do it on both junctions, that would be pretty strong evidence in my book.
Also use one primer pair spanning the entire insertion, which would not amplify if your long insert is present. This tests for heterozygosity.
yes John Schloendorn you are right but the thing is that we don't have the sequences of the inserted fragment because the company didn't provided it!
Sina Rahimi you can perhaps contact the company and they may provide the 20 nt sequence
Hello Sina,
I'm doing a similar experiment setup and I might run into the same problem. Did you confirm the knockout in DNA level?
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