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pcr problem


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6 replies to this topic

#1 siddharthsameer

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Posted 13 August 2012 - 02:33 AM

hello all
I have one question, i kept my pcr mix including my primer ,template with nuclease free water in the freezer at -20, and will amplify next day.. will it work???? or i have done a mistake icubating it, as taqmaster mix got finished and i have ordered it to make one more pcr.. kindly tell me i am in great tension here..
regards

#2 phage434

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Posted 13 August 2012 - 02:38 AM

It should work fine.

#3 ascacioc

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Posted 13 August 2012 - 03:36 AM

You anyhow you freeze all the components separetely for storage, don't you? So why woulnd't they work when they are together frozen? :) I did it before and it worked perfectly. Worst case scenario: the Taq polymerase in the mix will lose a bit of its activity.

Andreea

#4 siddharthsameer

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Posted 13 August 2012 - 05:19 AM

You anyhow you freeze all the components separetely for storage, don't you? So why woulnd't they work when they are together frozen? Posted Image I did it before and it worked perfectly. Worst case scenario: the Taq polymerase in the mix will lose a bit of its activity.

Andreea

thanks a lot but I think i will be keeping it for 2-3 days at - 20. waiting for my polymerase to come.. i hope it works....

#5 ascacioc

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Posted 13 August 2012 - 05:22 AM

Aaaah, you froze it without the polymerase: then there is nothing to worry about: I did it lots of times when I was doing like 4-5 PCRs a day of a few tubes each throught a week. This saves a lot of time. :)

Andreea

#6 siddharthsameer

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Posted 13 August 2012 - 05:38 AM

It should work fine.

thanks for ur reply but i froze it with polymerase, primer , abd my template, actually i had to do 4 pcr but eventually the taq master mix got over so I kept those 3 in freezer ...and waiting for the taq to come to make it for the another one.. do u still think i will be having proper amplification of my pcr.. m really nervous about that, and i think i have done a big mistake, already for the first time my pcr did not work...

#7 ascacioc

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Posted 13 August 2012 - 07:41 AM

Ok: I see a problem here, you froze Taq, template, primers and I assume water (no dNTPs?) without buffer? I usually add the things in the PCR mix in the following order: water, template, buffer, primers, dNTPs, Taq polymerase. I add the Taq polymerase last because you do not want to dilute the enzyme in water but in the buffer. On the other hand, Taq is one of the most stable enzymes/proteins I ever purified i.e. you need a lot to denaturate it and make it worthless. Now...if your PCR did not work last time as well, you can redo it from this master mix or throw it away and redo the entire thing. Depends on your situation: if that's all the template you had, than try to squeeze it out of there.

In the end of the day: it can work (most probably, if you did not do a big mistake the first time) or it will not work. You will know it only if you try. Otherwise, we can just talk whatifs until the end of time. I always say: while we talked here, you could have tried it already:)

Andreea




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