Any Universal Primer for 3' of cDNAs of all mRNAs? - (May/08/2008 )
Hi,
I have a human cell line which is infected with pDisrup containing neomycin insertion. I know for sure that one of the gene in this cell line has neomycin insertion. I want to analyse whether this cell line contains only one neomycin insertion or more in some other genes? I think if i get a common reverse primer for all mRNA cDNAs, i can get one or more bands of PCR product depending on number of neomycin insertions by using forward primer for neomycin.
It is a kind of alternative to southern blot which for some reasons i cannot perform at the moment.
can anyone guide me on this? I mean is there any poly A primer common for all mRNA cDNAs?
Your help will be appreciated...
Malik
In eukaryotes, almost all mRNA is tailed with a poly A tail. A primer TTT TTT TTT TTT TTT TTT VN is typically used to prime cDNA production.
It might be easier to do inverse PCR on your genomic DNA. Cut with a frequent cutter, religate, PCR with outward directed primers from your Neo gene, run a gel. You should do the religation in dilute conditions to favor circularization.
It might be easier to do inverse PCR on your genomic DNA. Cut with a frequent cutter, religate, PCR with outward directed primers from your Neo gene, run a gel. You should do the religation in dilute conditions to favor circularization.
Thanks for your suggestions. I will try this degenerate primer (i will use it as reverse primer) to amplify 3' end of mRNA cDNAs using my NEO as forward primer.
Thanks again.
Malik