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Can't get PCR with large overhang primers to work

PCR overhang low gc

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8 replies to this topic

#1 EtOH

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Posted 10 May 2013 - 04:03 PM

Hello,

I've been struggling with a PCR. So here's the deal. I have these 55-mer primers. 29 of those ntds anneal to my gene of interest and 26 are an overhang sequence I need to add on. The primers have the correct sequence - I've quadruple checked. A big problem is that my primers have a very low GC content. The Tm of the full-length primers is 68 and 66 and the Tm of the 29 bp that anneal to my gene are 42.5 and 45. I've tried all kinds of things - I tried a gradient from 51-61, I tried a gradient from 39-45 (I didn't want go lower because I'm sure I'd get lots of nonspecific binding). I also tried a gradient of 39-45 for the first 3 cycles, followed by 30 cycles at 61 degrees. Nothing worked. I got a couple of nonspecific bands at the 51-61 gradient and at the 39-45 followed by 61. I also tried adding DMSO to all of those reactions. I have some betaine but I'm not even sure whether to try it since nothing has worked so far. I initially tried the same primers but with only 18 bp annealing to the gene (b/c I was worried that I would get errors in the primers if they were bigger, but that didn't work either). There's no way I can change the sequence to a more GC-rich one. I'm using Pfu and the length of the gene is ~1kb.

A few more things I've done that have produced no change:
Increased template DNA conc.
Increased primer conc.
Increased annealing time
Increased extension time

Things I haven't done but I'm not really considering:
Altered MgCl2 conc
Altered buffer conc.
Altered polymerase amount

Some help would be wonderful!

Thanks in advance.

Edited by EtOH, 10 May 2013 - 04:17 PM.


#2 bob1

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Posted 10 May 2013 - 07:22 PM

Could you get some primers that are just the target sequence (i.e. no overhang) and test those, just to make sure that you can amplify the gene.

Did you make sure that the overhang is on the 5' end for the reverse primer?

#3 EtOH

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Posted 11 May 2013 - 12:33 AM

Yes, the overhang is on the 5' end and as of right now, I can't get the primers w/o overhang. Also I'm trying the exact same PCR (with the same overhang) with a total of 6 genes (all of which happen to be GC poor) and nothing is working

Edited by EtOH, 11 May 2013 - 12:35 AM.


#4 Makosad

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Posted 13 May 2013 - 03:40 AM

I have never done a PCR with that low Tm and to me it appears the problem.
Try with increased MgCl2 concentrations, if doesn't work, I think you have to make longer primers, with Tm around 60.

#5 phage434

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Posted 13 May 2013 - 05:00 AM

What is the GC content of the rest of the gene? You should definitely try again with a low EXTENSION temperature. I'd recommend 64 or lower. Annealng temperatures lower than about 48 have never produced anything for me, but very low GC cannot be amplified with a 68 or 72 extension. You need to extend for longer, of course.

See the paper by Su and Wellems:
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. No abstract available.
PMID: 8628694

#6 EtOH

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Posted 13 May 2013 - 03:23 PM

Thanks phage. The whole gene is about 40% GC but I'll still try lowering the extension temp.

#7 phage434

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Posted 13 May 2013 - 03:30 PM

All it takes to prevent amplification is a region about 25-35 bp long with very low GC.

Have you tried amplifying with Taq? It's not as fancy as the new enzymes, but it sure works.

#8 EtOH

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Posted 13 May 2013 - 07:40 PM

Soooo... It turned out that whoever labeled the ladders labeled them wrong, and the "nonspecific" bands I was getting were actually the correct product... And here I wasted almost a month thinking I can't get the damn PCR to work... Thanks for the help though!

#9 Makosad

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Posted 14 May 2013 - 11:46 AM

:D





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