Monthly Archives: July 2012

Lessons from the Lab Bench Part 1: DNA reprecipitation

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) 

Ingredients

  • 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.

Method

  • 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. 

Advertisements

Why angry loners are not autists, and Joe Scarborough is just plain wrong

Advance warning: this is a bit ranty, and more opinion than scientific. But please read it anyway. This is important. 

For anyone who has had their head stuck in the sand since Friday, on 20th June 2012 a young man ran into a midnight preview showing of the new Batman film where he released gas cannisters and opened fire with three guns, killing 12 and injuring 58 others.  James Eagan Holmes was in court yesterday for the first time to be charged with 142 counts of murder and attempted murder.

Continue reading

100 genomes of 100 year olds for $100 000

On June 26th 2000 the consortium of researchers working on the Human Genome Project announced they had created their first ‘working draft’ of the human genome sequence. It had taken a decade to achieve, with groups in at least 5 countries collaborating, cost $3 billion and provided 7-fold coverage (i.e. on average, for any single point on the genome they had seven sequences).

Twelve years have gone by, and you might think that we would be busy sequencing other things. (Well, we sort of are: bonobosbananas…) But actually we’re still looking at human DNA. We might know what humans look like at a molecular level ‘on average’: but on average doesn’t tell you why some people are more prone to cancer, or why some people are more likely to get Alzheimers, or why some people can taste PROP. It doesn’t tell you why some people can live to 100 either, in spite of having genes that make them prone to clotting disorders or dementia or heart disease.

The competition

In 2006, non-profit organisation X-prize announced that they would give a $10 million prize to the company that could sequence (and assemble) the entire genomes of 100 centenarians at a cost of less than $1000 per genome in 30 days. With an unprecedented level of accuracy. Given the HGP cost around $3 billion to complete, that’s quite an ask!

The general idea is that by comparing these genomes, particular area of interest might ‘pop out’ as being associated with longevity. 100 people is actually a pretty small sample size, but it’s a starting point and may identify several candidate genes that can then be studied by other (cheaper!) methods.

The first entrant

For the first time, technology has progressed to a place where achieving the aims of the X prize really is a viable possibility. The Ion Proton sequencer (which, with a bit of luck, we’ll be buying soon-ish – squee!) might just be up to the job, and as of this week its inventors are the first company to actually sign up to the challenge. There’s still almost a year left for other companies to get on board, but Illumina (makers of the GA II and the new-to-the-market HiSeq) have already come out and said that they don’t plan to participate. Oxford Nanopore and Complete Genomics are still not making any decisions about whether or not to enter: presumably because they’re waiting  to see what state their technology is in 10 months from now.

As per the good old $1000 dollar genome, $10 000 analysis script, most of the time isn’t actually going to be spent sequencing the genomes. The vast majority of the 30 days will be spent in assembling the sequencing fragments into longer strings that match the known order of genes in the human chromosomes: the biggest time-constraint for the competitors is getting the sequencing done as fast as possible to leave the four whole weeks for genome assembly. We’ve been hearing for a while now that the $1000 dollar genome is right around the corner, so it’s exciting to see Ion Torrent putting their money where their mouth is. Come next October we can see whether they’re right.

 

Grad student support networks: finding friends in unexpected places

The internet is now full of advice for new and prospective PhD students. Go visit your lab before you agree to work there. Talk to the other postgraduates to find out what the lab is really like. Start your reading before you matriculate, but don’t expect it to help you understand anything. Begin writing early. Make sure your support network is in place, and understands what you’re doing…

But wait a second. What support network? Sure, if you’re staying in the city where you did your undergrad, with friends and a boyfriend and maybe even family around you then that’s a silly question. But what if you’re moving somewhere new? What if you’re travelling cross country to a new city where you know nobody?

Continue reading

#summergoals: End of Week 1

At the end of last week I wrote a post about my #summergoals: aka the baking biologist’s attempt to be organised, and not have her life taken over by generic moping, procrastination, and political machinations. (Did I mention I’m an evil genius?)

As week 1 draws to a close, it seems sensible to establish how I’m doing so far (aka give myself a kick up the behind).

Continue reading

Molecular Biology 101: What’s an -ome when it’s at home?

Genome

Proteome

Transcriptome

Exome

Microbiome?

All of these words are becoming part of the scientific vernacular, and new ones are arising all the time. But is this proliferation of terms meaninful, or even a good thing?  Continue reading

My morning so far…

I’ve taken this off #Whatshouldwecallgradschool

The title is ‘When the -80 fails and I lose my samples’, which has happened to me, and was reacted to similarly to this. Today is less dramatic, but still requires a suitable amount of nerd angst. This is not helped by my having played squash for the first time ever, been suitably knackered this morning, and therefore turned off my alarm, gone back to sleep, and woken up at 8:40.

Le sigh.