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non specific amplification in clone fragment using primer

non specific amplification in clone fragment using primer - Protocols and Methods Forum

non specific amplification in clone fragment using primer - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 12-18-2006, 12:53 AM
muhammad yasir
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Default non specific amplification in clone fragment using primer



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.
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Old 12-21-2006, 01:14 PM
Jose de las Heras
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Default non specific amplification in clone fragment using primer


"muhammad yasir" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ]. net...


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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