non specific amplification in clone fragment using primer

Jose de las Heras via methods%40net.bio.net (by josenet At tiscali.co.uk)
Thu Dec 21 08:14:57 EST 2006


"muhammad yasir" <yasirphr At yahoo.com> wrote in message 
news:mailman.491.1166451422.19683.methods At net.bio.net...
>i use the pcr product of 16s rRNA and i am sure there is no other fragment 
>in this vector, but i am really supresied that amplification of the 1.5kb 
>16sRNA gene is preety good but getting one week extra band of extactly 
>double size 3Kb. Even i reduced the extension time to 1mints and increase 
>the aneling temp. now the band is more week but still exsist.


with most commercial Taq polymerases even 30 seconds is plenty to get 
substantial amplification of the larger fragments. Increasing the annealing 
temp shows a decrease of the intensity of teh second band, so that suggests 
a weaker priming site within the vector for one of your primers. Have you 
checked the sequence for close homology? Perhaps adding DMSO 5-7% might help 
improve specific annealing?
If you really want to know, you could always clone that extra band in a TA 
vector (pGEM T Easy, for instance) and sequence that directly, to see what's 
been amplified.

Jose 




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