1. That ladder suggests that you need to run the samples longer. It's not separating effectively. The gel size and the amplitude of the current also affect how much 'oomph' you need to pull the bands through the gel. Consider extending this time, if possible. This will also help you separate two sized bands in males (XY).
2. yes to the other comment on etoh extractions. phenol will kill your pcr.
3. where are you getting the reagents for the PCR reaction? if its a kit (or even ask for a free sample from some companies) follow their instructions if you aren't already. I've found them to be more robust in their guidelines than old papers (i assume its old since it lists the dNTP concentrations, and I don't see that anymore in recent papers).
For the Sigma kits that are linked on the wiki, these are hot-starts, and require something like a 5min denaturation to remove the antibody (or whatever it is in that kit) that prevents non-specific products from forming before the reaction 'should' begin. it's a great feature, but if you don't account for that in your pcr reaction, you'll get ambiguous bands... and none of the gels really convince me you/they have a product yet (on that wiki).
if my suggestions dont make sense, just let me know.
On Sat, Jun 30, 2012 at 1:30 PM, TRolandB <williambeaufoy@gmail.com> wrote:
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.- on the page I link to there's a section detailing our extraction protocol: complete protocol as of June 2012. - we use chelex.To view this discussion on the web visit https://groups.google.com/d/msg/diybio/-/xkgSsplQ_l4J.--
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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