dbell at fresno.ars.usda.gov
Wed Dec 11 11:30:40 EST 2002
We started out years ago using TaqPolymerase and then went to Taq Gold when it was developed (no promotional intentions here). Then there was a whole flood of polymerase enzymes being released that were less expensive, so we tried some of them. I found that each polymerase enzyme has its own "optimum conditions". Using conditions and primers previoulsy optimized for one brand or type of enzyme usually will not work using another enzyme.
If you are starting a new project, you need to screen primers, optimize [DNA], optimize [MgCl], and optimize annealing temps with the brand of enzyme that you intend to use throughout the whole experiment. Do not switch to another enzyme mid-stream.
I have also tried the REDTaq and the Extract-n-amp kit with very disappointing results. Extract-n-amp is a quick and dirty extraction where plant DNA is SERIOUSLY degraded, so it may be OK if you are doing PCRs that amplify short fragments. But for RAPDs, AFLPs, ISSRs (PCRs with larger fragments). . . it is not the way to go.
Just my 2cents worth, hope it helps!
:From: Gerd Nilsen [mailto:gerdn at ibg.uit.no]
:Sent: Wednesday, December 11, 2002 7:39 AM
:To: methods at hgmp.mrc.ac.uk
:I have recieved free samples of kits using REDTaq, at several
:occations. Sounds like a good idea every time, but it never works.
:Also other pople in the lab have tried REDTaq and gotten the same
:Still, the thing has survived in the market for many years, so there
:must be some ways of getting it to work:
:So, what's the trick?
:My last trial was with the REDExtract-N-Amp Plant PCR Kit. This is a
:kit for DNA isolation and PCR.
:To troubleshoot, I skiped the plant material and just added clean DNA
:and primers to the PCR kit. plant DNA (6 ng), 10-mer primer, 95 C
:denaturing, 35 C annealing (don't ask me why, I just stept into a
:ongoing procedure to test the new kit), 72 C elongation, 40 cycles.
:Works like a charm with Dynazyme, but not with the REDExtract-N-Amp
:Plant PCR Kit.
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