Week 3

So…this week I carried on with my rounds of PCR, gel running and gel extraction and finally finished this process, ending up with a final mutant construct that we’ve sent for cloning into a vector.

However, the process wasn’t completely problem-free! In one round I ended up with 2 fairly strong bands in one lane of the gel which was a bit confusing, so I ended up cutting out both of these and trying PCR with both of them as 2 different templates in the next round. This PCR didn’t really work very well and didn’t give particularly conclusive results about which band was the correct template, so we decided that I should run out a sample of the 2 extracted bands on a gel again to look at their sizes. Surprisingly, on this gel, they appeared to be the same size as each other – so I’m not quite sure what was going on the first time with the 2 very separate bands! As both of the bands seemed ok, I took what was originally the more intense one and repeated the PCR with this, which worked.  

Alongside this, I have been helping to purify out DNA from bacterial cultures for transfections. On Tuesday we used a manual method of plasmid prep which aims to give much higher yields than you can get with the ready-made Midiprep kits. However, the price to be paid for this higher yield is a very lengthy protocol! There are many rounds of spinning down for 10 minutes and incubating for 5 minutes, a step of leaving on ice for half an hour, lots of shaking tubes around to resuspend pellets...so it ended up taking the best part of a day to get to the stage where we left the pellet to dry overnight! Still, it’s worth it when you get such a good yield of DNA, as it means you should be left with enough for the rest of your experiments and so saves time later on.

Unfortunately, one of the samples didn’t give us a pellet at all, but we carried on with the other one anyway and picked some colonies for overnight incubation in order to yield some more cells for plasmid prep the next day.

So, the next day I was left to do a Midiprep – a much shortened down version of the manual method, but which yields less DNA. This went well enough – I managed to follow the protocol ok and didn’t make the same mistake as I did last week, which was to snap the end of a syringe off in the precipitator containing the precious DNA! (luckily my supervisor managed to rescue the situation by screwing in a nail and pulling it out)

On Thursday I set up several transfections with some of the cells we had cultured in tissue culture – each one had a different combination of mutant HDAC constructs with its binding partner. This involves incubating the correct amount of DNA (30μg) with the transfection agent, then adding this to cells and media to give an overall 30ml transfection volume – a bit of maths is involved to work out the volume of DNA needed and the dilution factor for the cells. Then each flask needs gassing for 2 and a half minutes before incubation – this can get a bit tedious by the time you reach flask number 6! My transfections actually worked well, meaning my supervisor could use them in immunoprecipitation at the weekend.

On Friday I had another go at the lengthy manual method of plasmid prep with the new pellets we had prepared, hoping that this time it would work. However…it didn’t. I got nearly to the end and things seemed to be going ok, but then unfortunately I lost any sign of a pellet being there. Oh well, I guess that’s just how science is sometimes! Luckily there wasn’t an urgent need for this DNA, so we’ll probably repeat the transformation with a new culture and try again sometime next week – 3^rd time lucky hopefully!

The disappointment of the failed plasmid prep was soon forgotten when we went bowling after work. I hadn’t been for years and had never actually tried it with the barriers down, so I was a bit worried I’d end up with a zero score…but I actually managed to surprise myself and did ok in the first game. By the second game (after a few glasses of wine…) my luck/skill deteriorated a bit! It was all pretty good fun, and a good end to a busy week!