So I’ve been trying different methods to clean up DNA for the last week. I tried polyyethylene glycol (PEG) precipitation followed up with ethanol precipitation – quite a long process but quite straight forward. To do this involves lots of waiting and lots of centrifuging. After leaving the DNA/PEG mix in solution overnight, you then centrifuge at 13 000rpm the sample for 15 minutes after which you are meant to see the DNA form a pellet. As my yield was low I couldn’t actually see the pellet, so I had to ‘guess’ where the tiny invisible pellet might be (theoretically at the bottom of the tube on the side). Then you have to do the entire process again with ethanol/sodium acetate in a further cleaning step. The lab-tech and myself had no idea if all of this effort had been worthwhile. Thankfully the nano drop showed that 50% of my samples showed a better 260/230 result (basically the beetle DNA was ‘cleaner’). My yield was OK but quite low. To improve the yield I then just tried the ethanol step alone as less steps means you save time and DNA. This gave similar results which was pleasing.
Did this mean that my PCR (the DNA replication step) worked any better? It did, but it’s still only worked on some of the specimens, even ones that had a good nano drop reading. Frustrating. I then went back to the literature/supervisor to see what I could do next. I could either re-extract using a different technique (and still have the same problem), or use the ‘Rolls Royce’ of taq (Multiplex PCR) and add some extra things (BSA and Q solution) to the reaction. My hypothesis is I that detritus/soil off my beetles is interfering in the reaction (see this paper: http://www.sciencedirect.com/science/article/pii/S0038071706000678).
Hopefully this is the case and I get more consistent success…..