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Reason for odd PCR conditions


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3 replies to this topic

#1 mcb56

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Posted 29 December 2010 - 09:21 AM

Hello, I am running a PCR with the following conditions: 94C for 1 min, after initial denaturation, 94C for 15 sec, 55C for 30 sec, 72C for 30 sec, repeat 15x then 94C for 15 sec, 55C for 30 sec, 72C for 45 sec, repeat 25x.

I am amplifying off of plasmid DNA a 600 bp product. I am curious as to why I have one set of steps (repeated 15x) with an elongation of 30 sec and then another set of steps (repeated 25x) with an elongation of 45 sec. I am using Taq polymerase. The PCR works great, IO just don't understand the need for 2 sets of steps.

Thank you

#2 atwood

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Posted 30 December 2010 - 08:22 AM

actually, i think 1 step is ok~ i also dont see there is any reason for separate it into 2 steps
longer elongation time just for synthesize longer DNA fragment~

May be this is a special technique (although i dont think so). Let see how other ppl answer :unsure:

#3 Maddie

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Posted 30 December 2010 - 11:02 AM

I've never seen this either. Where did you get this protocol?
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#4 hobglobin

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Posted 30 December 2010 - 11:56 AM

I saw this type of PCR in a kind of mixed touchdown and gradient protocol. But then in the first part the annealing temperature is decreased with every repeat. The second part is then the actual amplification of the fragments that could be synthesised in the first part.
This is e.g. used in cross-species amplifications (primers of different species are tried out for a new one).
Anyway perhaps you overlooked the temperature decrease?

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.





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