Tuesday, December 9, 2014

False economies

So how’s the stagiaire, you might ask, that girl with the unfortunate timing and worse training ?

We were going to look at the clinical data for her samples before deciding which of them to choose to analyse, but ran into the problem that she doesn’t have with her, and cannot get access to, the clinical data (the f*? can’t someone email her a list? – is there no list?) So we have to be satisfied with the labels already assigned.

The problem of how to pick which ones to analyse has been very simply resolved. It turns out that most of the 300+ samples she brought with her are completely degraded and cannot ever be analysed. Only about 60 actually contain DNA in fragments long enough to do anything with. So we’ll go with the 60, whatever they are.

300 blood draws, and 240 of them are good for the trash? What happens when you don’t make the necessary investments up front, but try to get by as cheaply as possible. Instead of using some modern but relatively expensive kit for extracting the DNA, they used the tried and true phenol-chloroform method.

When I was in grad school, a wise post-doc always said “The fast way is the slow way, and the slow way is the fast way.” Very good advice, that. Same can be true of expensive and cheap, when cheap puts success at risk.

Now, you can get acceptable DNA with this method, but you have to really know what you’re doing. This is the method I used way back when I was a tech, and it was not always a success. The first thing to do was to get your reagents right, and that meant spending all the necessary time under the fume hood buffering the phenol to a neutral pH. No neutral pH, no good DNA. Just forget it. Whatever comes out might look good at first glance, then fall to pieces over the next week or month, leaving nothing.
Once you’ve got your phenol neutralized, and you extract the washed white cells from your blood, recover the aqueous phase, re-extract that twice with chloroform to get the phenol gone – and a third time if needed. Yes, just do it again. Phenol is the kiss of death for any further manipulation. Chloroform just evaporates no problem. Now you can precipitate your clean(ish) DNA with ethanol and just a bit of salt if there isn’t enough already. But don’t spin it down. Nice DNA will form a beautiful, fluffy white cloud, like magic, as soon as the ethanol hits it. Seal the end of a glass pipet (ooo yes, get to play with fire in the lab!), and fish the blob of DNA out (do let the pipet cool before dipping it in – otherwise the ethanol at 60% will ignite). Squeeze the DNA blob like a sponge against the edge of the tube and once the ethanol has evaporated, swish it around in a tube of aqueous buffer and let it resuspend for a couple of days before attempting to measure its concentration.

So our student is working on the samples that can be worked on, and we’ll see on Monday what sort of results can be had. Cross our fingers that once she passes the preliminary amplification step, all the rest is ok. After all, the rest all works off of the product of the first step, not the primary material. Because if there are no results, um, well there really is no plan B.

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