PCR 3kb fragment - (Aug/28/2006 )
I'm trying to PCR a 3kb+ fragment. As the results are negative despite I tried optimising and changing DNA polymerases, I decided to chop the fragment into two: i.e., I plan to separately PCR the 1st half and the 2nd half of the cDNA, then only join them together.
May I ask how do you design the primers in this case? Must some region from the reverse primer of the 1st half overlap with the forward primer of the 2nd half? And in this case, do I need to add in RE sites in order to join the fragments?
Your suggestions are highly appreciated =) THanks for helping a noob!
Are you sure you have a full length transcribt in your cDNA? (and how?)
How is the cDNA generated (pT-primer, random-hex-mer or gene spec. primer)?
Which kit or RNA-DNA-polymerase is used?
yep the prob is i'm not sure if i have full length transcript.
the cDNA library was generated by my supervisor =\ not sure how he did it coz he aint around now.. but i suspect maybe the cDNA quality is not good
i used Clontech's Advantage 2 PCR Kit, Expand Long Template from Roche, and also regular Taq (promega).
Try to establish a rt-PCR for the 5' + 3' end of your transcript, products should be around 200-300 bp. If there is an 5'UTR, put at least the 5' forward primer in there. May be you can use DNA as positive CTRL and the -RT-batch of your cDNA preparation as negative CTRL.
I am not familiar with the clontech kit, but the Expand kit from Roche works fine (as any 'long template' kit, I guess. It is may be not the best, but good enough. I have amplified DNA streches of 12 kb with this kit, but this is of course also template depending).
Unfortunatly, there is nearly no way to test the quality of your cDNA, except by testing spec. transcripts. You can test for expression of GAPDH (or any other so called housekeeping gene), but this will only tell you, that the RT step has worked, but not how good (I guess, you have already done this test, as we all work accoring to GLP ).
But if the RNA was fine (checked by gel) nearly all RT-Kits work fine (more or less).