PCR question

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unibas
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PCR question

Post by unibas » Sun Mar 06, 2011 10:35 am

what happens if after the first denaturation and annealing steps, I take the tube out of the PCR machine and leave it at room temperature for an hour or so? would the primers remain stuck to the template?
if not, how can I make sure the primers remain on the template for about an hour?

this is not a theoretical question, my experiment depends on the answer.

thanks.

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DRT23
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Post by DRT23 » Sun Mar 06, 2011 11:40 am

Since lower temprature makes DNA-primer binding more stabile and while annealing temprature is about 50 C and room temprature is 25 C, the primers should remain stuck to the template. But, if you want to be sure, you may compare this procedure (removing the tube after the first annealing step and keeping in room temprature for an hour) with the regular procedure by using a quantitative-PCR. Starting with 2 tubes that are identical in terms of amounts and types of ingredients, if primers remains stuck to templates in your procedure, there will be same amount of DNA products at the end of amplification, after same number of cycles for both procedures.

unibas
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Post by unibas » Sun Mar 06, 2011 12:35 pm

thanks! I'll do that.

follow up question: suppose the primers remain bound after one hour at room temperature. after this one hour, is it possible to release the primers from the plasmid? I guess if I just heat it to 95 and cool it again, the primers will get back on the plasmid. but I don't want the primers anymore at that point.

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JackBean
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Post by JackBean » Mon Mar 07, 2011 12:57 pm

you could increase the ionic strength or cool it slowly, than it will take enough time and rather longer chains should anneal.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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