December 15th, 2006

StichAtWork

Lab Diary (this week)

Here's what I did this week:

*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.
*Sequenced.
*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.
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Dandelion

(no subject)

We had quite a windstorm last night, even this far inland. Our house, exposed up on top of the hill, was moaning and groaning all night, and the wind blew rain (horizontally) into the windows so hard that it looked like a car wash...just sheets. I haven't seen winds that strong since the storms on SAS. No real damage here...one of the big maples by the cemetary came down across the highway last night, but they got it all cleared away by this morning. The power flickered a lot, but never went out.

Here's hoping all my friends in Seattle are okay. My Mom's not answering the phone...she's probably picking up branches in the backyard. Anyone remember the 1993 Inauguration Day storm? I was in 5th grade, in Ms. Higgins' class. I rode the bus to Briarcrest that morning and actually saw a Douglas fir come down onto somebody's house and garage on the way. I was at school for a few hours, and we could see trees falling outside the window. The power finally went out in the middle of the ceremony (we were all watching it on TV as part of our unit on goverment), and they sent us home in the middle of the day, which was very exciting. There were branches and debris everywhere. The power was out at my house too. Dad and I got the gas stove out of the attic and Mom made us hot chocolate by the light of the propane lantern.

It's been a busy few days. My semester effectively ended last Thursday, with my last class and a final presentation and homework in PopGen. We had a Secret Santa gift exchange in the herbarium on Friday (I got a little fountain), a full-on Herbarium party at Pam's house the next day (I made grilled zuchinni), and then Kim came over on Sunday to hang out. She gave me some lovely ornaments and some green origami dragon earrings (wearing them now), and we watched Happy Feet and The Incredibles.

I'm very much looking forward to going home soon. :)
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