Hi,

You could add DMSO or betain to your PCR reaction. This might help in case of high GC templates.

Moreover, there are polymerases with a special high GC buffer available e.g. Q5 (NEB). You could try this polymerase instead of your Taq. Another advantage of the Q5  is the proofreading activity of this enzyme.

Good luck!

yes, Mr. Boas Pucker you are  right DMSO can  use for high GC templet 

I made good experiences with the Phusion polymerase from FINZYME. It has proofreading and can handle tricky sequences. One should use proofreading polymerases for clonings. Template conc from 50-300 ng and sometimes higher is totally okay. You should check that your primers don't cover SNPs which might destroy the primerbinding to your template. You can send me the primer sequence if you need help.

Dr. Kim is right about Phusion. I'm a big fan of it for cloning as it has high fidelity and can combine the annealing and extension step which can be useful in high GC. They also provide a high GC buffer which has been helpful for me in the past and is not so expensive that it can't be use for all cloning projects, thus reducing the issues of mutations created by standard Taq. 

Yes, William H. Broach is totally right about the GC buffer. First, one can use the HF buffer (high fidelity) which is optimized for minimal error rate of the polymerase. But if this didn't work out, I tried GC buffer (higher error rate than HF buffer, but better performance in GC rich regions).

Thank you all for the suggestions.

Dear Broach & Kim: I have noted to use the Phusion enzyme and GC buffer for the future cloning assignments, till then I have to troubleshoot with the reagents which we currently have. I have PCR amplified other GC-rich promoters with the same Taq PCR kit NEB (E5000), so i think I should try all other means of troubleshooting, then for the next slot I would get the recommended enzyme and buffer. I tried by altering the primer (0.4 -1 uM) and template DNA concentration (10-30 ng/ul), changing the source the template DNA, and changing the annealing and denaturation time from 30 to 40 seconds. Also I set one reaction with 2% DMSO, as recommended by Pucker. For DMSO, set I got no satisfactory result, but for others I got a very high intense high molecular (~1.6 PCR band). Only in one case, I got the desired band (~693 bp), but that also accompanied with two high molecular intense bands and a kind of smear.

Attached is a photo of the gel where I run PCR of three promoters with different conditions :

Lane 1: 1 kb ladder

Lane 2: PCR band (desired) [32 ng/ul DNA, primer concentration 0.4 uM, annealing and denaturation 40 s] [Thermo long PCR kit]

Lane 3: 100 bp ladder

Lane 4 & 5: no result: primer dimer at the bottom.

Lane 6: 100 bp Ladder

Lane 7 & 8: desired PCR band [12 ng/ul, primer 0.4 uM, annealing and denaturation 40 s] [Taq PCR kit NEB]

Lane 9: blank

Lane 10: desired band between 600-700 bp (~693 bp) but high mol intense bands (primer 1 uM, DNA 30 ng/ul, annealing and denaturation 40 s) [Thermo]

Lane 11: No band (Primer 0.4 uM, DNA 32 ng/ul, DMSO added, annealing and denaturation 40 s)[Thermo long PCR]

Lane 12 & 13: non specific high mol band (primer 0.4, DNA 12 ng/ul, annealing and denaturation 40 s)[NEB]

The Tm of the primer is 64.37 and 64.61 degree and annealing temp is 58 in all the cases.

checking your primers, the primer binding sites contain SNPs (common SNPs 147), so they can be a reason why in the HeLa cell line, no pcr worked.

You could take templates of other human beings and do the same pcrs, if such differen PCR creates the desired band, then the probability is very high that the HeLa cell line differ in the bases of at least one of the SNPs. (from which source was the DNA template for line 10?)

Dear Kim,

Thank you for the suggestion. All these are from the HeLa DNA. DNA is of very good quality both in terms of purity and concentration (kit isolated). The reason for choosing HeLa is because it is a common cell line and also carry very less mutations. But the promoter I am working with is new so there is no prior work with this which I can make a reference point. But the good thing is that I have few more DNA sources from different tissue origin. So, before changing the primers I bettee use them and if I still face similar issues, I will redesign the primer. 

according to your primers, the expected product size (without restriction sites etc) is 693 bp.

none of your shown bands is exactly at 700, rather below 500 (line 7 & 8) and below 600 (line 10). None of the product is the desired product (you will see that, if you sequence the products).

When I was cloning stuff, I was like you and took bands of 'similar size' as successfully amplified products - but with time again and again I realized that this gives bad surprises. So after several years doing lots of PCRs I am very strict with the heights of bands in agarose gels. I can tell you the bad news that none of the bands in this picture will contain your desired product - sadly.

Dear all,

I thought to end the query with a satisfactory answer. In my case, the problem seems to be in the genomic DNA. We repeated the entire protocol of cloning with the kits to ensure the pure yield of the plasmid and genomic DNA. Although the approah seems to be a bit expensive but it saves lots of time and effort.