Plan for validating cDNA clones portal 1-7-13

2015-01-13

azim58 - 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&amp;shva=1#sent/13c1252974b3c939

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