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Difficult sequences in PCR

I cant get the rig

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2 replies to this topic

#1 Red-eyed Rabbit

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Posted 24 October 2011 - 02:15 AM

Dear All,

I have a problem again. I'm trying to make a PCR, but inside the product (so _not_ the primer region) containes a difficult sequence, I just call it "repetitive-like sequence", because most parts of it consist of 2 nucleotides, but not exactly a dinucleotide repeat, just almost (sth like this: tatataatattatat...cacacacccaacaca....gagggaga of course each "unit" is longer). All my reactions before and after this seqence are working, only this one is not, though I have tried several primer pairs, including a forwrd of the 5', and a reverse of the 3' product from this sequence
example:
nnnnn...P1F...nnnnnn... P2F .....nnnnnnn.....P1R ... atatataattatattttatataaaatatat.. ....P3F......nnnnnnnn P2R ...... nnnnnn..... P3R.....

P1F-P1R: ok
P2F-P2R: not working
P3F-P3R: ok
P1F-P3R: not working
P1F-P2R: not working
P2F-P3R: not working

we tried with DMSO and temperature gradient, result is the same. I tried several different primerpairs, too, so "P2F" and "P2R" refers to several primer pair, not only one, and from different distance (thus different amplicon-size) from the problematic sequence.

Do you have any ideas how can we overcome this sequence? sadly, we really need this part to be amplified. Any help or idea is welcomed.

#2 hannna

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Posted 24 October 2011 - 03:48 AM

What DNA Pol are you using?

For me Phusion Pol works much better than Taq with difficult sequences.

#3 phage434

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Posted 24 October 2011 - 04:20 AM

Lower your extension temperature. Very high AT regions bind so poorly that at 72C the 3' end of the extending strand will not extend. I'd recommend extending at 63C for double the time normally used.

Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA.

Su XZ, Wu Y, Sifri CD, Wellems TE.
Nucleic Acids Res. 1996 Apr 15;24(8):1574-5. PMID: 8628694




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