Wednesday, 20 June 2007
My New Editing Regime...and Memories of Subcloning
The publisher and I have agreed a deadline for Joshua bk 1 v3.0. I'm deep in the process of writing Jaguar though, and can't let the momentum go. So I try to work on Jaguar in the morning at my desk, take a two-hour break to refresh and then it's on with the editing, which seems to require a different skillset as far as I can tell. Thank goodness for editors. I've said it before and I'll say it again. Mine is probably going to save me from being a laughing stock, if nothing else - hopefully a lot else but you can't predict these things.
I like to take my manuscript out for little walks. I can't be bothered going all the way to the Bod this time around - I'm only spending 2 hours a day on it, what with the Jaguar writing taking up all my morning brain activity. So I've been going to Summertown. The above photo is taken of my set-up at the Summertown Wine Cafe, a bijoux little joint on South Parade which makes the best coffee in Summertown (there are many Italian coffee machines in Summertown, but few baristas who have a clue how to use them). Sadly however, they charge a small fortune for savoury food - best to stick to cake, I'm trying to avoid blimpdom so that's out. Blah, blah, blah. Nothin of consequence in this entry sadly. I'm just writing something to have to test in a new way to do an RSS feed. If you read this, you've just participated in an experiment. Do you feel used? I kind of miss doing experiments. Somewhere in the back of my mind is the niggling feeling that a PROPER day's work is what I used to pull off at the height of my keenness as a graduate student...a long day in the lab which ends with a successfully identified new DNA subclone to use in a lovely biological experiment.
'Subcloning' is a way of starting with a widgey little bit of DNA that is no use to anyone and two days later having bucketloads (as much as a milligram!) of the stuff that you can use to do biological experiments in tissue culture cells or even in unsuspecting fluffy creatures. (Some journals are so fussy that you can't get published unless your results are in a live organism.)
You insert a piece of experimental DNA into a 'vector' of usually bacterial or yeast DNA which has the ability massively to replicate it. Then you can grow the 'bugs' in a 500ml culture overnight and in the morning extract enough DNA to 'transfect' cells which allow you to test the properties of your experimental DNA. The tricky bit is that when you try to stick your experimental DNA to the vector DNA, only a small fraction will combine to give you the subclone. The rest will just be vector DNA that sticks back to itself.
When I were a lass we used to pick at least 24 bacterial colonies in the hope that 2 or 3 would have the subclone. It could take up to a whole day, a day spent 'doing minipreps', as we used to call it. Sometimes you had to use radioactivity and horrible, ooky, gloopy, neurotoxic polyacrylamide gel to help identify the subclone.
(Any molecular biologists reading this, bright young things with your PCR, your DNA synthesisers and sequencing machines...it's all very easy now, I'll bet.)
But! Throughout most of career as a molecular biologist I noticed that although I was a good little scientist and picked my 24-48 colonies everytime I wanted to find a correctly subclone, more often than not, colony 1 (the first I picked with a sterile toothpick) actually had the subclone. i.e. I didn't need colonies 2-24 and all the effort in 'working them up' was not actually essential.
Other people in my lab noticed this too. It turns out that in maths the number 1 is disproportionately represented (there's some rule and it's used as a way to detect fraud), well, in molecular biology this seems true too.
Don't think we let that observation go to waste, either. Towards the end of my time in the lab, I would often just pick a colony right off, inoculate my 500ml flask and grow up the bugs without testing whether they had the subcloned DNA in them. It saved a whole day! Of course I tested a sample before I used it to transfect my tissue culture cells. Well, duh.
If you didn't understand a thing I wrote in the last few paras, tell me. R1X did, so I have tried to rewrite it so that it makes sense.