*Ran a PCR on a selection of samples using my new primer (TrnK).
*Poured and loaded gel. Results unacceptable (ghost bands, lots of non-specific binding).
*Begged Cort for some GoTaq, which he kindly gave
*Ran PCR again with GoTaq.
*Poured and loaded gel. Worked fantastically...better than ever.
*Ran Exo-SAP-it reaction.
*Ran PCR sequencing reaction.
*Did ethanol precipitation.
*Joined and cleaned up all 13 sequences that worked.
*Sequences were all identical...no polymorphism at this locus at all. Start over with new primer.
*Did research on especially polymorphic primer loci from the Tortoise and the Hare paper.
*Recorded five that looked good.
*Went through the freezer and scavenged. Found a pair that were on my list as good.
*Looked up the PCR protocol. Annealing temperature was given as a range, not a discrete number.
*Found out that the thermal cycler has a feature that can run temps on a gradient, so I can pinpoint which temp is best for my samples.
*Set up and ran gradient PCR.
*Poured and loaded gel. Discovered that reactions work much better when you load them with GoTaq POLYMERASE, not GoTaq BUFFER. -___________-
*Did PCR over again. Is running now.
And if it works, I'll decide which gradient temperature is best, and do a PCR with a spread of my samples like before (about 16 of them), sequence them, and find out if this locus is polymorphic enough to be interesting (that is, polymorphic at all). And if not, I start over with a new primer again. And if so, I start pushing all my samples through...all 200-ish of them.
Important lesson: if the thermal cycler is booked up, ask people nicely for some space. This is the second day in a row where it's been totally full, and other people have moved their stuff for me with no fuss at all. Very cool.