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smears instead of bands


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#1 flashboy

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Posted 23 June 2004 - 02:27 AM

hi guys,

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!!!!

#2 SVTX

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Posted 05 August 2004 - 09:04 PM

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?

SV

Edited by SVTX, 05 August 2004 - 09:04 PM.


#3 preeti

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Posted 06 August 2004 - 09:42 AM

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
preeti




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