no product after 5' Race - (Jun/01/2006 )

Hi everyone,

I want to amplify the cDNA end of a partial gene. It worked for 3' Race but not for 5' Race. I have checked the quality of RNA, expression level of the gene in total RNA as well as the primer that I used before I start. A clear band (same as the expected size) can be found after first strand cDNA synthesis. I was then gene clean the single stand product and adding a poly-A tail by terminal transferase. However, no PCR product can be found after the first round of PCR reaction with my GSP and Oligo dT-anchor primer. Can anyone of you giving me some suggestions for that?

THANK YOU FOR ALL OF YOUR ATTENTION!!!

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Which kit did you use?
Why would you expect a band after the first round of PCR? Or did you mean after 30 rounds of PCR?

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I was using RACE Kit from Roche. I can find a band with expected size after cDNA synthesis (which is reversely transcribed with use of gene specific primer). After the first round PCR (35 cycles), no product can be found. I have tried to do the nest PCR (35cycles again), smear with size smaller than 200bp can be found, that is much smaller than the expected size (should be around 700bp). Would you mind giving me some suggestions on that?

Thank you very much!!!

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I'm having problems with that kit too. Did you perform the PCR control reaction? In my case it seems OK before the tailing reaction, but not thereafter. I'm not sure if the problem is the tailing reaction or the 1st PCR.

I used BD Clontech's kit before that, but results were very similar. I'm now considering Invitrogene's Generacer, but I don't know if it's any better than the other two I tested...

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