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Failed to amplify a 2.1 kb PCR product


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#1 june

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Posted 22 February 2005 - 09:35 AM

Hi, guys:
Does anybody can help me? I am trying to use the PCR to get 1.7kb and 2.1kb different PCR products, none of them works, I didn't get any PCR products at all. I used the Pfx, pfu and also high defity Taq from Invitrogen as well, the PCR is 94c 45sec, 60c 1min, and 68c 2min and 35cycles. something is wrong there? please help me to slove the problem.

june

#2 Simonsays

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Posted 22 February 2005 - 11:29 AM

I don't know the lenght of your oligos, but they must be pretty long if your annealing temperature is 60C. When a PCR doesn't work, the first thing I try is to lower this temperature (I even lowered it to 35C). The chance to get non specific amplification is greater, but it might work. Otherwise, I performed denaturing at 95C (94 should be fine), and elongation at 72C (you do 2 minutes... you could add another 30 seconds, since Taq does about 1kb per minute).
I also always finish with a 10 minutes step of elongation at 72C in case there's some unfinished amplification.


Simon

#3 labprincess

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Posted 22 February 2005 - 07:47 PM

Hey,

You could try a touch-down reaction rxn. It tends to be a bit more forgiving with regards to your primers. Also increasing your extension time a wee bit could help.

Hope that helps

LabPrincess :D

P.S. I love High Fidelity Taq , it is sooo much easier to deal with than pfu :)

#4 Scott

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Posted 07 March 2005 - 08:41 AM

Hi,

The first thing I would do is check the melting temp of the primers and start dropping the annealing temperature down. If that doesn't work, it will get messy by checking your components in your kit. How old are the dNTPs, what template are you using (could this be the problem) etc. Really shouldn't have problems with MgCl concentrations for most basic PCRs.

Regards,

Scott




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