So basically I got really excited about the ITC. It came out with some amazing results, which I really wanted to prove. So, I was on a roll, the machine was working, and I'd done everything I could with the various bits of my proteins. I decided to (well it was my supervisor's idea really) try full length protein, and a mutation that had been made for something else. I could work out why when I ran these proteins I ended up with no heat exchanges (NOT good) and a really weird curve, that no model could fit.
So anyway, when I took the liquid out at the end of the experiment, it wasn't clear (as it should be), it was cloudy- the precipitation. That explains everything.
So after some discussion, it has been suggested that I change the salt concentration. Unfortunately, I've run out of these proteins (loads of the other ones) So I have to make some more. So, this week, I'm going to be learning how to do protein extractions. So far, I've just transformed the plasmids into the cells, using heat shock.
(Jargon here is...) Plasmid: a piece of circular DNA found in bacteria that are not part of the nucleus.
Transform: get the plasmid to get in the cells. The cells are E. Coli cells. To heat shock them, you leave them on ice for a while, then quickly heat them up to 42 degrees, then cool them again, before allowing them to grow up. So you know you've only got the cells with the plasmid in (that contains the DNA that codes for the protein) the plasmid has an antibiotic resistance gene in it, so that when you grow them in some broth with antibiotics in them, only the cells you want survive, and the rest die because they can't grow in the antibiotic.
I will write with what else I'm doing later in the week. For now, I'm happy to be doing something new, great as the results are from the ITC, you're relying on a computer, which is an amazing piece of technology, but I LOVE being in the lab. It's one of the things I'm enjoying actually. I really like the diversity of things you get to do every day!
No comments:
Post a Comment