Plan+for+validating+cDNA+clones+portal+1-7-13

-Design primers that will amplify from the poly T tail to the beginning of exon 1. I would then do PCR on the tumor and normal samples. If I see a difference in tumor and normal I will know these transcripts are different between the two. If I don't see a difference this will not really prove anything since the tumor cells may have the normal transcript in addition to alternative transcript(s) -Amplify the transcripts using the polyT+gene specific primer on the 3' end and the 5' cap primer with the kit. I would use the PrimeStar polymerase (other polymerases may also be tried like Dynazyme and Taq) that Andrey and I have observed is very resilient and can yield extremely long products (from our vector linearization work). I would then clone this gene specific cDNA into a vector (using pJET cloning) and sequence several transformed clones to see if the genes are truncated, contain frameshifts, contain the expected mutations for those with mutations, etc. --2-11-13 Update to this plan. I think I will have two sets of cDNA construction. One set with SMARTScribe and Advantage and one set with PrimeScript and PrimeStar. I'm not sure if I will have enough 5' primer that came with the kit though.

see e-mail thread with Kathy https://mail.google.com/mail/u/0/?ui=2&shva=1#sent/13c1252974b3c939