So I am pretty much completely obsessed with Tina Turner. I'm planning a post about the parallels between TT and Maria Callas. I don't use the F word lightly, but they are both so fierce. (I will have to watch Mad Max: Beyond Thunderdome yet again.)
But, after all, I do need some frenetic TT to get me through the last week in the Cone lab, which shaped up to be a non-stop shit-show, cluster-fuck. I was trying to finish up some last experiments so that I wouldn't have to get my coworkers to find my 4.5-years of unorganized supplies while I am 2000 miles away. So, we did finish most of the field work (I think) and I am finishing up the last experiments as we speak. So to say.
I have slept 5 hours in the last 48, but for some reason I am still going strong. Here's why: I've found that by staying out of the daylight, I can totally subvert my body's natural circadian clock. I actually caught myself purposefully not looking out the window at work today so that I would fool myself into experiencing perpetual night.
I know. That is so vampire-emo.
One of the last experiments I am doing is a real-time RT-PCR, which is a pretty powerful (if not standard) genetic technique. Basically you make cDNA from a RNA sample, and then carry out PCR with the cDNA to quantify the transcript level. Sounds pretty simple right?
In theory. But of course as Homer Simpson likes to say, "In theory communism works."
So you have to flash freeze your tissues in liq N2, grind them, extract them with trizol (that helps keep the RNases from chewing up your nucleic acids), chloroform extract the trizol, precipitate the RNA with isopropanol and salt, wash the RNA with EtOH, and resuspend it back up in DEPC H20. After each step you have to centrifuge, and remove the supernatant with a Pasteur pipette.
To keep from getting the reverse transcriptase from extending any genomic DNA contamination, and therefore negating your RNA quantification, you have to then digest your total RNA sample with DNase I, then phenol:chloroform: isoamyl-alcohol extract, and resuspend.
Then you need to carry out the RT rxn with MMLV (monkey murine reverse transcriptase), RNase inhibitor, dNTPs--then PCI extract the first strand cDNA, use spectrophotometry to determine absorbancy, and then run real time PCR in technical triplicate with endogenous controls to quantify RNA levels. I won't even go into the primer design phase, but those have to be gene-specific and optimized separately.
Ideally you would assay the integrity of your RNA and then check for inhibitors of the qPCR rxn. On a formaldehyde (denaturing gel).
The real-time PCR uses florescence to quantify the accumulation of double stranded products in real time; by including a passive reference dye and SYBR green (which binds to double stranded DNA) you can measure product after each of the 40 cycles.
I know. Pretty ridiculous, right? Considering that I didn't know how to do any of this about 5 weeks ago, I think I am doing OK.
Considering that I've barely started on manuscript preparation, I have to somehow copy most of my lab notebooks from the last 4.5 years, and take my shitload of files.
Did I mention that I am leaving on Tuesday morning?
I plan on packing Sunday and Monday. That will be fun and exciting.
Go westward young man.
It has finally hit me that I am starting a completely new life in a state 2000 miles away where I know a handful of people. I will miss Craig and zipper so much! Not to mention the other Columbia friends L&K, Rachel, etc.
Sorry that was totally rambling, and probably mostly incoherent, but I am about to nod off into a coma for the ages, so gentle reader...goodbye for now.