We're performing a long range PCR, longer than 3kb and so far it has failed to work. We've tried DMSO, Mg2+, touchdown approach, different DNA, new primers, different polymerases.
I'd appreciate any suggestions.
run the pcr in final concentration of 1 molar betaine ( sigma very cheap). It helps the template to melt so improves pcr particularly for high GC templates. Works with all polymerases
Hi,
The LongAmp Taq (NEB) worked very well for PCR products up to 15 kb in my hands. Have you tried this enzyme already? However, most polymerases like Q5 or Phusion are able to produce products > 5 kb.
Best,
Boas
Thanks Boas! I've tried Phusion and Q5, but didn't get anything :) I'll give it a go with a LongAmp. Would you recommend any other tiny tricks?
Most important is the quality of your template. Have you check your DNA e.g. via NanoDrop? The ratio of 260nm/230nm should be high (at least > 1). An increased amount of dNTPs could be beneficial for really long products, but I do not have any strong evidence for it.
You can also try AccuPrime (Thermo Fischer). It can amplify up to 5 kb. The high fidely one can amplify up to 20 kb.
I'm also trying PrimeSTAR® GXL (Takara) for >20 kb.
I think we can do more suggestions if you show us the protocol that you have used for each enzyme. But like Boas said, the quality of your template is of utmost importance. You need also to check the amount of enzyme and Mg ion in your reaction. And evaluate your cycling conditions.
3-10Kb is not so too long,if you can not get your products,you should not only thing about the polymerases,maybe the other reasons,
1) Are you sure your template is working?is it right ? you can test it use other genes;
2) Whether your sample is high GC contents ? If so ,use high GCbuffer or polymerases;
3) Is you PCR primers right ? or the high anneal temperature ? you can make a gradient.
4) Whether the nuclesae pollute ?in your water or spearhead ? you can use some news;
Hope the helps ! ! !
run the pcr in final concentration of 1 molar betaine ( sigma very cheap). It helps the template to melt so improves pcr particularly for high GC templates. Works with all polymerases
Hi
I used TaKaRa LA Taq ® Hot Start Version polymerase for amplifying 9 Kb and it worked very well.
You can try this with different PCR cyclic conditions.
Good luck
260/280 really doesn't tell you everything about the quality. The DNA could be heavily degraded or sheared and you won't see that by NanoDrop. Run on bioanalyzer or a gel to gain insight into whether DNA is intact. Definitely amplify a shorter product of the same gene. For example, I'd keep the FWD primer the same and then order a couple different REV primers that would result in different size products; say maybe 800 bp and 1500 bp. Good luck and keep us posted of your success.
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