I am using the the Transfer PCR protocol by Erijman et al. (2011) and have had success in applying the technique to a number of cloning efforts.
I must say the technique/protocol is very powerful, but there isn't much information on the subject, when one runs into a problem.
I am having difficulty with the first part of the PCR, where the gene to be transferred is not being amplified. The gene is rather high in GC content (67%) and as such I am using the GC buffer that comes with phusion. After trial and error, I have amplified the gene by increasing the number of cycles from 13 to 30 and the primer concentration rather high (~200 nM).
1) I don't see any problem with having high number of cycles. Unless having low number of cycles is important for the entire procedure?
2) The high concentration of primers is apparently not good for integration into the recipient plasmid, so I am wondering whether I could purify this pcr product by gel extraction and continue onto the second half of the Transfer PCR.?
3) An additional problem that I encounter is that the primers are likely to amplify a certain region of the recipient as well as the gene of interest (unlucky gene sequence I guess). This refers back to question 2). ? If gel extraction in between the two PCR cycles is detrimental to the transfer reaction, could I at the least run the 1st part of the PCR without the recipient plasmid and then add it in the second half?
You can refer to the folowing link to get some more details:-
http://link.springer.com/protocol/10.1007%2F978-1-62703-764-8_7
Yeah, I have that problem sometimes, too, and when you're cloning you don't have the option to simply move to a less GC-rich region. Consequently, I ahve done a lot of PCR trouble-shooting. First, I think using the GC buffer is a good start. A few other ideas:
o try different concentrations of DMSO (maybe 2, 4 and 6% final) in your amplification
o Have you tested different annealing temps?
o 13 cycles is very low...25-35 is more common, 40 is fine (though by the later cycles, the dNTPs become increasingly spent).
o I assume you mean 200 uM (micromolar) primers, which is about right. You can try up to about 500uM, but note that higher concentrations will lower your annealing temp.
o If you have more than one produce, you can try gel-purifying it. There are a bunch of kits out there. I am partial to the Zymo Research products (for example: http://www.zymoresearch.com/dna/dna-clean-up/zymoclean-gel-dna-recovery-kit), but there are many good options. After the gel-cleanup, you will have no troubles with the next PCR.
I can give you more advice if you like.
Sandip, thanks for the reference. I had the paper to set up the reactions. The problem is that this paper doesn't really go into the crux of my question.
David, Thanks for the comments regarding GC-rich sequences. The 13 cycles and the low (nM) concentration of primers are specific to Transfer PCR.
My main question is whether it is okay to gel purify the megaprimer produced by the first reaction and then use it for the second reaction?
Ah, I see. I have not read up on transfer PCR per se. It sounds like the low primer concentration might be to avoid contamination of the second PCR. I just took a look at the abstract and my guess is that you would be fine doing a gel purification before the second reaction, but, given my unfamiliarity with the details, you won't want to rely too much on my advice.
Still, I would say, give it a try, especially if you can distinguish success or failure of the second reaction by agarose gel.
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