There comes a point in every woman’s life when she realises that the world is expecting her to have developed some sort of culinary prowess, and she’s not quite sure she’s caught up yet. It’s the time when the world seems to think that ‘take the carcass of a roast chicken’ is an acceptable way to start a recipe, and you’re left wondering at what point in the 5 years of vegetarianism that covered the ages of 19 to 24 you were supposed to have learned how one actually roasts a chicken? It’s when a friend tells you that making macaroons is just like making meringue and you have to admit that your last attempt to beat egg whites within an inch of their life ended with your mother-out-law turning up while you were up to your elbows in batter and you still haven’t quite recovered from the experience.
Being a postgrad in a molecular lab can often feel rather similar. Especially if you’ve come straight from undergrad, while working under a postdoc who had five years experience in the lab before he even applied for a PhD. There’s a certain kind of terror that arrives with the question “CTAB, Phenol-chlorofom or high salt?” when accompanied by an impatient look and the expectation that you know what any of those things mean.
Two months into my PhD I did DNA extractions for the first time, and was expected to have a favourite method. None of these nice Qiagen kits or TRIzol reagent: I had to make buffers and solutions and figure out molecular weights and I was supposed to have a secret method tucked in my lab book that had been passed down from PI to postdoc to PhD like my grandmother’s recipe for Christmas cake. (Which is, incidentally, very good). And what’s worse, there didn’t seem to be any single resource I could look at or an explanation of how these methods differed or where they had come from or why we used them.
A week ago I had a similar experience. Having done some gDNA extractions for the first time in over a year I needed to clean the stuff up. (Horrible 260/280s). I googled DNA cleanup and found kits for cleaning up PCR products. I googled DNA purification and got extraction kits. So I asked the Twitter hive mind (#phdchat) and low and behold, help was forthcoming. I had to try a few different things, but I now have what appears to be a really nice re-precipitation protocol, which I shall put here for any and everyone who encounters the same problem. And you’ll forgive me, I’m sure, if I write this out recipe style!
DNA Re-precipitation (aka clean up aka removal of isopropanol)
- 100% ethanol. (No, you don’t need the super-pure VAT-ed stuff. Stick it in the -20 to keep it ice cold).
- 70% ethanol. (Yes, 70. Not 75. Not 80. 70%. Also in the -20)
- 3M Sodium acetate at pH 5. (This is a pain to make, so leave yourself an hour to dissolve it before you need to use it. If you have a magnetic hotplate, today is the day to use it. And don’t forget to leave sufficient volume to allow you to stick a shedload of HCl in it to drop the pH – although I’m told it still works at pH 7 – when you first make it it will be ridiculously alkaline, and no good for anything.)
- Your DNA sample.
- Make sure your sample is in a tube where it fills < 25% the volume (e.g. a 2 mL Eppendorf can take up to 500 uL). Personally I’d pick a 1.5 tube, because then even if you can’t see your DNA you know it’s in the corner
- Add one tenth of the volume of Sodium Acetate (e.g. add 20 uL to a 200 uL sample).
- Add two volumes of ice cold 100% ethanol (i.e. 400 uL)
- Freeze it for ten minutes to an hour.
- Set yourself a timer for 50 minutes to remind you to turn on your centrifuge so you can get it down to 4C.
- Centrifuge samples for 15 minutes at top speed and 4C.
Pause to be completely confused and start panicking. You were expecting a tidy white pellet weren’t you? Ah no, today you can have a huge glistening gooey smear. Don’t worry: it’s full of DNA. Try to get the supernatant out without sucking up the goo and you’ll be fine!
- Remove supernatant using your p200.
- Add 200 uL of cold 70% ethanol.
- Centrifuge for 5 minutes at top speed and 4C.
- Remove supernatant using a p200.
- Leave to air dry. (Pro tip: when it’s completely dry it will also be completely clear. Don’t freak out when your pellet disappears.)
- Resuspend pellet in 20 uL water.
Et voila! Perfect DNA.