SCIENCE WOES
Sep. 17th, 2008 11:15 am![[personal profile]](https://www.dreamwidth.org/img/silk/identity/user.png){kind=small}
gnommiHoly Hell, Batman... This RT-PCR stuff boggles my brain. Well, not the theory, but you know, actually making the damn thing GO.
I *think* I have my gene sequence, mRNA sequence, sizes etc. I understand the principles of primer design (no hairpins, Tm considerations, length, yaddayaddayadda). I'm hoping that someone in the lab can point me at a primer design program and walk me through it cos none of the interfaces I've looked at are even remotely intuitive. THEN of course I have to do the actual factual RNA extraction (RNEasy kit?), reverse transcription and then the de facto PCR bit. Run the gel, look at the size of the product band and Bob's your uncle? I'm guessing that there are kits for most of this but probably budget-busting.
All I want to do is to verify data I already have about protein expression from FACS and immunohistochemistry (and some wobbly Western Blotting). My protein is present at very low levels by the looks of it, but I can't say that it's unequivocal... and yeah actually a lot of my thesis hinges on knowing for sure either way.
ARGH.
You see why I might want to take up glassmaking/beadweaving/plumbing/farming/taxidermy/barmaiding/marrying a millionaire instead?
I *think* I have my gene sequence, mRNA sequence, sizes etc. I understand the principles of primer design (no hairpins, Tm considerations, length, yaddayaddayadda). I'm hoping that someone in the lab can point me at a primer design program and walk me through it cos none of the interfaces I've looked at are even remotely intuitive. THEN of course I have to do the actual factual RNA extraction (RNEasy kit?), reverse transcription and then the de facto PCR bit. Run the gel, look at the size of the product band and Bob's your uncle? I'm guessing that there are kits for most of this but probably budget-busting.
All I want to do is to verify data I already have about protein expression from FACS and immunohistochemistry (and some wobbly Western Blotting). My protein is present at very low levels by the looks of it, but I can't say that it's unequivocal... and yeah actually a lot of my thesis hinges on knowing for sure either way.
ARGH.
You see why I might want to take up glassmaking/beadweaving/plumbing/farming/taxidermy/barmaiding/marrying a millionaire instead?
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(no subject)
Date: 2008-09-17 01:12 pm (UTC)so things that would be simple like "what tubes do i need, where do i get primers, how do i make primers, what shouldn't i do, and what should the end result look like" all need hours and hours buried in textbooks, research papers and websites.
and of course, then the end result is directly proportional to how well you researched how to do it.
and before you even get to this stage you have the "what do i want to know and what techniques are available to find out this information" bit.