(no subject)

[sammyphoenix]

This stuff never seems to work out for me. I am half way through collecting my time points and I have been trying to run a gel on them. Now, usually I break open cells and purify the protein before I run a gel, but since I'm trying time points to get an idea of when protein expression is highest, I must use a very crude method.

So, I spin down the time point cultures (1mL each), giving me a pellet of bacteria that may or may not be expressing protein and the old liquid media. I dump off the media into waste and add loading buffer directly to the bacteria. This loading buffer has some nasty stuff in it that will break open some of the bacteria, boiling also helps bust open the cells, and I have to boil anyways because that denatures the proteins so the dye in the loading buffer can bind to the proteins.

Then, comes the tricky part apparently. I have to spin down this cultures again. Because the bacteria cells have been broken open a sticky mess is made within the tube. This sticky cell gunk does not like to be pipette up. Spinning at a very high speed will separate the cell gunk from the proteins release from the bacteria. Main problem here was that not all of the cultures spun down at the same rates, making me have to load my gel half hazardly, and have some of my samples sitting in the gel, which usually isn't a good thing, while waiting for the others to separate properly.

And of course when I put the gels into their holders it didn't seal properly, so the running buffer leaks out of the center chambers. So, I have to stop the electrical power running the gel and add more buffer every 10 minutes to make sure the gel runs evenly. If I let the buffer get lower than gel top the electrical current can run through the gels properly and my protein bands (if I have any) will be in a convex arc rather than an even line (since all my samples are the same protein).

I just think science/work hates me. Maybe I wasn't meant for this job or something.