April 19th, 2001


(no subject)

Today was another nothingish sort of day. I went to work, and did stuff. The PCR I did yesterday didn't work - but I talked to Christian about it (as he's the resident PCR guru ;) ) and he had some useful thoughts and suggestions. So I'm trying one of them out tonight (well, I set it up at about 4pm and it's in the machine overnight). Hopefully that'll work ... though if it does it suggests that I've got contamination in one of my primers :( I should probably back up and explains. I'm doing mutagensis using an overlap extension PCR method. Basically I do 2 PCR reactions on bits of the DNA that overlap at the bit I want to mutate (with primers that have the mutated sequence). Then you do another PCR using the outermost primers and the 2 products of the other two PCRs, and that gives you the full length mutated cDNA. So I'm stuck on the first step - the initial 2 PCRs. One of them seems to have genomic DNA in (not just my plasmid template) and the other produces products that are bigger than expected (about 10 times) and smaller than the template (about half the size). The first problem is what I'm looking at with this test PCR tonight - I'm using one primer from each reaction (everything else is the same in both) so depending if there's genomic contaminant or not I'll know which primer is the problem. The other problem is a little harder to overcome - it may be that the mutagenic primer I'm using in that reaction binds somewhere in the vector DNA so I get lots of different products :( I'll have to wait and see (and use the computer tools to check where the primer binds in the vector).

That won't have made a lot of sense to most other people reading this but whatever ;) it's what I did today :)

I also had a meeting with Gill today. Partly about work stuff. Partly she seems to be reorganising and shaking things up a bit. She had a little chat about 'time management' which from the way she said it wasn't actually directed at me (private meeting, just me and her, but she said 'If I'm saying this to some people I'm going to say it to everyone ... '). I'll probably move bench - back through to the other room, and next to the people working on the same stuff I'm working on. Which will be quite cool - my PhD project was quite far removed from what other poeple are doing in the lab, but now I'm working on the TIMPs there are a fair number of others working on the same sort of thing :)
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