Thanks to all for the advice. In reply
- The ladder always works, so no problem with the electrophoresis I would think.
- The fragment sizes are 977 and 788bp, quite large fragments I think?
- Good idea on the multiple PCRs, we may give that a go.
- Good idea on the multiple PCRs, we may give that a go.
- on the page I link to there's a section detailing our extraction protocol: complete protocol as of June 2012. - we use chelex.
On Friday, 29 June 2012 01:35:16 UTC+1, cory....@gmail.com wrote:
The first step would be to really figure out which step is the
problem. Have you tried doing multiple PCRs on the same DNA
extraction?
My suggestion would be to do 3 different DNA extractions all from the
same person. Then do 3 different PCR reaction on each sample, mixing
the PCR reagents separately for each reaction, then (assuming you are
confident that your thermalcycler is consistent across wells) run all
the reactions at the same time and then load them all into the same
gel side by side. From there you should be able to tell if there is
variability between extractions, between PCRs, or both.
Also, what exact protocol are you using for the extraction? Your wiki
links to a couple of different protocols. If you're using
phenol/chloroform I would suggest doing multiple ethanol
precipitations as phenol inhibits PCR.
-cory
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