Two months later and something to show for it

In October, I was troubleshooting and developing microsatellite methods.  In November, I was troubleshooting and developing microsatellite methods.  In December, I was troubleshooting and developing microsatellite methods. In January… Oh wait, did I mention I got them to work?   I wrote earlier that I thought trying new TAQ polymerase would help amplify microsats and, after obtaining it along with more microsat primers, I was able to get very nice amplification of almost all of the microsats (except, ironically, the ones I had been troubleshooting with).  Because JumpStart TAQ is more expensive than my standard TAQ I’ve been using, I experimented with the recipe a bit more and was able to get good quality amplification with this TAQ for some of the microsats and have since been busy with PCR amplifying each DNA sample twice for each microsat.  So far, I am ‘done’ with coq-211 and coq-20.  The next fragments in line are coq-27, coq-208, and coq-221.

Also, I developed a hybrid protocol for running DNA-PAGE (DNA polyacrylamide gels).  This was a humorous process because the people I asked about this process thought the whole time I was trying to run a western blot for proteins and told me to use the ‘right’ buffers for gels and running buffer (they were right if you want to run proteins…when used with DNA, the samples float out of the wells in the gel with horrifying speed and diffuse across the running buffer like an oil scim.  Horrifying.)   With some luck and Google, I found a non-denaturing protocol that uses TBE buffer for both gels and running buffer.  I’ve used this buffer in the past with agarose gels and so I knew this would work…except there are a few general things to keep in mind when running DNA-PAGE.  1.  Higher concentration gels solidify faster and are better to use if you have a leaky caster set. 2.  Run multiple markers because if you tend to get impatient and try to run the gel at 75 volts instead of some lower voltage like 65, the sample bands run crooked.  3.  About 30 minutes before you are done with the run, turn the voltage way down to 30 volts.  This will crisp up the bands and you can see the heterozygotes more clearly (if you are running microsatellites and are particularly looking for variation such as this).  4. Try VERY CAREFULLY to remove the gel from the spacer plate after staining in order to visualize it under UV.  This makes a much cleaner image.

So as I was saying:  In January, I am traveling with my research adviser to Hawaii Island to collect more frogs which I will bring back and begin the whole process of DNA extraction and PCR hopefully with significantly fewer hitches.  I may be crazy when its over, but I can’t wait!   I  got my first data yesterday and it only took a year and nine months…

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