RT-PCR a GC Rich (68%) gene (2200bp) - (May/12/2006 )
I'm trying to clone a gene (2200bp) that is GC rich (68%) using RT-PCR. 'm using invitrogen's taq polymerase high fidelity. Using cDNA and primers specific to the gene I ran the following rxn: 94C at 2minutes, 94C for 30 seconds, 55C for 30 seconds, 72 for 3minutes (35 cycles) and extension at 72 for 10 minutes. I did not get a single band, any ideas would be really apreciated
Different magnesium concentraions might also help.
Increase the annealing temp.
Use different enzyme specifically to amplify GC rich regions.
Its kind of difficult to suggest any one thing there are lots of variables.
Good luck !!!!
how is the quality of your RNA and cDNA? if you are certain that's good, and you are using the correct annealing temp and such, I would say that Phusion (NEB polymerase) has a buffer specifically for difficult/GC-rich templates
I have not used it for that purpose, but for easier templates it's an excellent polymerase and I have had pretty good luck with it
"Not getting a single band"...
Is that a) not getting anything (not even a single band)
or multiple bands?
In case a): try to check the quality of your cDNA/RNA, in case optimise your PCR (mostly Mg/annealing temp).
I haven't done it for RT-PCR, but betaine or DMSO really helps with high-GC standard PCR. I've gotten good results on 85% GC viral DNA, so 68% shouldn't be out of the question...
Make sure your enzyme is not a "hot start" enzyme -- they require 15 minutes at 95C before they are active. If you are getting no bands, try lowering the annealing temperature. If you have a gradient cycler, use a gradient cycle for the annealing temperature, from 48 to 64C. Redesign of the primers would be high on my list if the problem persists more than a day or two. Primers are cheap, and your time is not. With this GC percentage, I would be adding DMSO or Betaine at the 3-6% level.
Could u tell me the RT conditions(temp)? U have to use 5-10%DMSO, so that it can take care of GC rich areas or secondary structures.