SCIENCE WOES

[gnommi]

Holy 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?

Comments

[wrong1.livejournal.com]

Maybe I'm still too fresh faced and new to it all to be jaded yet, but that actually all sounds really exciting.
I'm champing at the bit for my third year proteomics stuff.

[gnommi.livejournal.com]

it's exciting until you get THE FEAR
whatifidoitwronghowwilliknowthenwhatinthevivaARGHwhathappensifipressthisbuttondoestheworldexplodeETCETC

mainly the bit that is annoying me is how un-intuitive all of the gene databases are. I clearly don't sprechen geneticsch yet, 'cause a lot may as well be Greek! In fact, thinking about it, it actually might be.

I am very much in favour of buying an RNEasy extraction kit and a one-step RT-PCR kit at the moment. Pretty funky tech: shove dNTPs, enzyme/buffer mix, primers and template in a tube all at once, shove tube in thermal cycler, go make tea whilst SCIENCE does itself!

Alternatively, it's intern time of year. Maybe I should go and harpoon a couple and harness them to my data-making machinery. MUAHAHA

[wrong1.livejournal.com]

Yeah, I can imagine. The project I'm doing basically involves running assays of cell proteins to analyse the ratio between phosphorylated and non-phosphorylated proteins. If I manage to do the quantitative assays ok, then I get to do some qualitative ones and if they go ok, then I get to run both over the 2 life cycle stages of Chlamydia, which is essentially new science and may get me published as a collaborator if anything good is found.
Obviously, I'll be putting a lot of pressure on myself to get that done as that would be CV gold, and would potentially give me a very easy pathway into carrying ont he research as a PhD in the new centre for proteomic research they're building.

So yeah, while all of the techniques sound like fun to me now, I bet they'll scare me witless when I actually peg my hopes on how well they turn out.

[gnommi.livejournal.com]

it's also nice that you get someone to show you how to do it, once you are on a PhD you are largely on your own.
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.

[squidflakes.livejournal.com]

You don't have EasyBake Oven: For Geneticists on every street corner over there? I'm shocked!

[gnommi.livejournal.com]

gief!