9/19-9/23

This week I:

Located BSA in the lab and protocols/reagents necessary for salt-out DNA extraction.

Continued to edit my personal and previous research statements for the NSFGRFP.

Identified my topic for the NSFGRFP proposal essay:  This will focus on examining the rate of accumulation and occurrence of deleterious genotypes in hybrid tiger salamanders.  It was shown previously that the hybrids exhibit a large degree of phenotypic diversity, but so far, no studies have examined in depth the occurrence of deleterious genotypes, although they are expected to exist.  Broader implications of this proposal address the use of hybridization in species conservation.

Contacted and heard back from 2 potential advisers at Florida State and heard back from 1 potential adviser at Cornell.  Advisers at both universities are accepting students in the fall and I am continuing communication with them.

Finished (again) my primary lit presentation for November.

The Woes of PCR

Since coming back to the fall semester at Jewell, I have:

1.  Verified that my PCR reagents are in good working order

2.  Run PCR for 16S ribosomal RNA subunit (mtDNA) from kit-isolated and saltout-isolated DNA from coqui frogs

3.  Run PCR for microsatellite coq-28 and coq-31

4.  Run PCR for microsatellite coq-31 under the following conditions:  annealing temperature gradient (49-55 degrees C), 2 uL template DNA (versus 1 uL) + temperature gradient, 1.0 uL MgSO4 (versus 0.5 uL) + temperature gradient, and 2 ul template DNA + 1.0 uL MgSO4 + temperature gradient.

So far, only 16S works (and it works really well).  The next step is to include BSA (bovine serum albumin) in the PCR mix.  Apparently that will coat the inside of the PCR tube and make the reaction work better.  Some say it’s magic…in fact I know of at least three people who swear it’s magic.  If that doesn’t work, I still have room to increase Mg, which will increase binding but lower specificity, and decrease annealing temperature, which also decreases specificity.  If all else fails, I will try ordering the mix the authors used for $45.00 from Sigma.  (or someone else will, and I’ll just ask really nicely).