I am trying to generate a ~100 bp amplicon from a very GC rich region (76%). My PCR will work one week then fail the next. I have tried using other primers and I have run my gDNA out on a gel. I have some mild degradation of my DNA. I store my DNA at 4C, extract using a Qiagen kit and elute in buffer AE. My PCR is failing in samples that don't have mild smearing in the gDNA gel. I have been trying to get this to work for a month. I don't know what to do!
Dear Elisabeth
i doubt your problem is your gDNA; rather the fact that your loci is approx 80% GC rich. This is what I would do:
- add 10% DMSO to your PCR and/or 1M betaine
- Commence your PCR with an initial denaturing cycle of 96C for 5 min
- During your PCR denature for double the usual time and denature at 98C
- To compensate heat inactivation of your Taq add double to 3 x the recommended amount
- better still use a highly processive resistant polymerase like phusion where you can routinely and stably denature at 98C
- If that doesn’t work end your primers in 2 xGC residues and if your primer is very GC rich and has a melting temp of 65-70C perform a 2 step PCR in which you denature at 98C as mentioned and both prime and extend at 65C
- amplify for 35-40 cycles
Dear Elizabeth
It's difficult to point out the exact reason, so I would try series of PCR troubleshooting. For example, what tissue do You use for extraction? I know that some "tissues" like feces can contain PCR inhibitors, also knowing 260 to 280 and 230 ratios will be helpful. Also, CpG-reach primers could need higher annealing temperature. Also, I usually avoid to put several C or G in a raw at the beginning of the primer, I do not know for sure, but I red once that it can lead to some problems. Well, it is just beginning, so let's close these points.
Dear Elizabeth, the attached file may be helpfull for your study. There are some suggestions for PCR Troubleshootings.
Best regards.
Dear Elisabeth
i doubt your problem is your gDNA; rather the fact that your loci is approx 80% GC rich. This is what I would do:
- add 10% DMSO to your PCR and/or 1M betaine
- Commence your PCR with an initial denaturing cycle of 96C for 5 min
- During your PCR denature for double the usual time and denature at 98C
- To compensate heat inactivation of your Taq add double to 3 x the recommended amount
- better still use a highly processive resistant polymerase like phusion where you can routinely and stably denature at 98C
- If that doesn’t work end your primers in 2 xGC residues and if your primer is very GC rich and has a melting temp of 65-70C perform a 2 step PCR in which you denature at 98C as mentioned and both prime and extend at 65C
- amplify for 35-40 cycles
You can visit this page. I hope it can help you in troubleshooting. Good Luck!! http://www.bio-rad.com/en-kr/applications-technologies/pcr-troubleshooting#gel3
I am using Accuprime GC rich DNA polymerase and adding 2% DMSO to the mixture. My gDNA is from HCT116 colorectal carcinoma cells and I extract using the Qiagen kit. My 260/280 ratios range from 1.96-2.03. I am following the recommended denaturation time of the kit. I have gotten the reaction to work occasionally but cant get it to work consistently
Dear Elizabeth
Thanks for clarifying this. What is 260/230 ratio?
Assuming that it is good, next points that I would check are:
- PCR mix preparation (do You use pre-made or prepare by yourself)
- Efficiency of the enzyme (try from another band)
- Temperature of the primers annealing
- Increase amount of primers
- Increase DMSO up to 10%
I am sorry if I listed very basic things, I am sure You are aware about them, but just to be safe that most likely things are covered. For example, once my inconsistent PCR was caused by adding 0.5uL primers individually.
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