5' RACE problem - (May/09/2001 )
Hi, everyone! I have been using 5'RACE to get the unknown 5'end of my gene. I use a Roche kit for this purpose. Unfortunately so far I have not got any success. I synthesized cDNA using SP1 (gene-specific) primer, after purification and tailing, I performed first round of pcr using a polyT-anchor primer and SP2. I got two similar large band and a few smaller and weaker bands. using the first total pcr product as template, i performed the second round of pcr using anchor primer and SP3, the above mentioned bands are still there but stronger. i gel extracted the two larger bands, and verify them with different primers, then strange things happen. When i use anchor and SP3, as used in the 2nd round of pcr above, i got several bands but not one as expected; when i use a pair of gene-specific primers, i got a band identical to a control pcr product in which a known part of the gene was used as template; when i use ONLY the anchor primer, i got a band similar to the size of template; when i use ONLY the SP3 primer, i also got a band similar to the ctrl pcr. i ever cloned the band and sequenced it, it is totally another gene, no any similarity to my gene. I don't know how this could happen. i gave up the smaller bands because of their unreasonable sizes. If you know the reasons, please tell me immediately. a lot of thanks in advance. Cao.
I think the problem is the cDNA you synthesized. I suggest you use mRNA instead of total RNA and optimize your synthesis time.