RACE artifacts - (Mar/09/2005 )
I have cloned the same truncated 3´RACE fragment using two different RACE kits. I obtained them after two rounds of PCR with a lot of cycles. Now, I am trying to quantify the transcript in several tissues, but I obtain the same band in the negative control (without the retrotranscriptase) than with cDNA, and so, I think that I have genomic DNA in my samples and that the 3´RACE fragment could be a PCR artifact.
Could someone help me with that?
Thank you in advance!
i did 5' and 3' RACE-PCR on two genes before. in my experience, you should use NESTED primer to make sure your product is real one. by the way, if you are not sure the quality of your RNA (the starting material for RACE ) you can purchase high quality RNA (without DNA contamination) from CLONTECH, or even 5' or 3' RACE-READY cDNA. good luck!