smears instead of bands - (Jun/23/2004 )
i have successfully run pcr reactions looking for ucp-2 mRNA for 4 years, however, in the last months the pcr has stopped working. instead of easily repeatable bands, i am getting no bands and just a smear down the whole lane.
i use abgene's pcr reddy mix mastermix tubes which have always been fine, and reverse transcribe using 10 units of RT which again has always been fine
i get some more success with my actin bands, this led me to believe it was my ucp-2 primers which were the problem [as positive control was not working either] but some actin bands also disappear, and now my positive control has started to give bands again..... HELP!!!!
Hey I am also having a similar problem, but with genomic DNA. It always works the first time, quite beautiful band. But then, the reactions stop working. Just like yours a smear down the lane upto 1000bp or so. Even the negative has the same smear. Where do you buy your primers from?
i am posing this problem too
i tried changing primers(to qiagen .inc) and renewed all reagents for PCR,got some success
also take care all this are RNase free b4 u start working