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5'RACE

primers High Tm

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#1 LpBio

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Posted 05 August 2012 - 02:26 AM

Hello people,

I have been using GeneRacer [ligase-mediated] Kit in order to amplify the 5' region of a gene of interest (~200bp). The problem is that for both initial and subsequent nested PCR the designed and kit's primers have a high Tm 76/78 oC. The polymerase i'm using is Platinum high fidelity which suggests max. 68 oC as annealing temperature and 68-72 for extension. Should I exceed the 68 as annealing and use up to 71-73 , and what would the efficient extension temp be?


Thank you a lot.

#2 Trof

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Posted 05 August 2012 - 06:38 AM

Tm could be calculated by many different methods. The GeneRacer primers have Tm about 64-69 when calculated with Primer3 algorithm, which is the one I mostly use and have experience, that annealing usually works best -4 degs from this Tm.
So AFAIK you don't need to go higher than 68.
Try the recommended conditions in the manual first. As with any other PCR, you have to find the optimal annealing or use touchdown (which is part of the protocol if I remember well).

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#3 LpBio

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Posted 06 August 2012 - 08:26 AM

First of all thank you. Secondly, the problem is that I designed them according to the simple equation 2AT+4GC, as suggested in the manual, so now I have the following pairs of primers with Tm [calculated by Primer3] :
initial PCR {designed} 62 - 69.4 {kit} ΔΤm= 7.4
nested {designed} 73.9 - 64.9 {kit} 9

and I see their Tm difference is big enough.. So, is it preferable to design new ones or use the lowest Tm as annealing temp?

Posted Image any advice welcome..

#4 Trof

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Posted 06 August 2012 - 12:24 PM

Maybe because primers are quite cheap, you can try to design another gene specific primers with primer3 (so that the ΔΤm would be less than 2) and while you wait for delivery, try the primers you have. I think maybe it's better anyway to have more primers to try in RACE. If you start with touchdown you can even have both of them in the same run.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






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