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big differnece in my primers Tm

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#1 zogene



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Posted 19 January 2012 - 02:25 AM


I have to do cloning and I have cDNA but the problem is with primers, I designed the primers and eventually one of them has Tm 66 the other is 80, I did gradient PCR with control DNA and I have very small barely seen band at 76, can i proceed my experiment though or should I redesign my primers?

any advice will be appreciateed.
Thank you so much in advance.

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#2 Trof


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Posted 19 January 2012 - 09:55 AM

Tm 80 is really high. "At 76" means what? Barelly seen band would be difficult to clone. I would redesign.

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#3 hobglobin


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Posted 19 January 2012 - 10:48 AM

As it's a gradient PCR 76 surely refers to 76°C as one of the annealing temperatures (as lowest?). Anyway I'd redesign it too, for me around 72°C is kind of limit (then having a two step PCR) and the differences anyway shouldn't be too large.

Edited by hobglobin, 19 January 2012 - 10:49 AM.

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