Wednesday, 27 July 2011

Flasks, smells, and mess!

Right, I promised to try and not start my posts with so, and the last one was.  This one I'm struggling already.
In the past two days, I've been preparing cells.  After getting my plasmid to stay in my E. Coli (well I hope anyway), I grew the cells on some agar with antibiotic on overnight, which I did by spreading the mixture of cells over some agar.  You make agar by mixing growth media (broth) with agar, which is a gelling agent.   You can put pretty much what you want in the growth media according to what your cells need.
The next day, I nicked 4 flasks off the shelf that had LB in (this is the growth media that we use, it smells a bit like marmite diluted down, and looks like a urine sample), but apparently E coli love it!!! When I say nicked, they are there to use, we have a make some more if you take the last ones, and as I made some that day as well that was alright.
In these flasks I added more antibiotic, and some of my cells.  You just scrape a few colonies off your plate and ping the pipette tip into the liquid.  Gilson pipettes work by displacing air, and you use a new tip that fits on the end for each measurement, and they have an ejector mechanism, for each tip, so the tips really do ping off.  The advantage of this is that the cells are sticky, so aren't very easy to get off anything the attach to, so it's easiest to just put the whole thing in!
I also made 16 big flasks.  These each hold 500ml, and were double strength LB.  That's a LOT of flasks!
The little flasks I grew overnight, then added a bit from them to each of the 16 big flasks, and grew those up (in an incubator that shakes them to get air in and keeps them at 37 degrees) for two hours before adding 20% arabinose, which apparently gets the cells to start making the proteins.
Then I spun them all down, hence most of the smells and the mess.  You have to pour the cells into bottles, then balance the bottles, so the centrifuge doesn't go walkabout (like your washing machine does, but the middle goes at about 8000 rpm, so you can imagine how bad it would be!), and then tip the liquid off after spinning, and add more cells until you've got through all the flasks.  The cells smell, and are really difficult to keep all in one place.  I used plenty of sodium hypochlorite (the active ingredient in bleach) today to get everything clean.
Prepared loads of pipette tips for autoclaving (the big ones go in coffee jars, a good gauge of how much caffeine you need to be a scientist!) whilst I was waiting for my cells to grow, along with random helping people whilst they dashed off to do all sorts of other things.
Will update on if I actually get any protein from all this!!! :D

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