Interesting article by Corey Bradshaw about Google Impact Factors and ecology http://conservationbytes.com/2013/11/18/hate-journal-impact-factors-try-google-rankings-instead/. Ecology letters, as you would expect, rates highest (83) but Biological Conservation (58) and Conservation Biology (57) rate 2nd/3rd respectively. This is interesting as traditional IF rates these particular journals much lower (~4.3). Even more interesting is that PLoS One has more impact than Ecology Letters with and Google IF of 131. This isn’t surprising as it’s an open access journal as more people can access PloS articles so are therefore more likely to cite them. It is however another good reason to publish in journals that make science free to the world audience.
After a month of PCR trouble shooting I’ve finally worked out a methodology that will work consistently for my specimens! DNA purification, as I have mentioned previously, worked inconsistently so I turned to PCR additives and Qiagen Multiplex Taq . Following by the protocol developed by Thormann et al (a great paper by the way – see below), I initially had success with my staphylinids (including Atheta TFIC sp 01 below) using the 28S D3 marker. No surprises there as this was what is the primer set was designed for. However I was surprised by its consistency – every specimen worked perfectly even if the DNA template wasn’t good. I was possibly a little too excited to see nice clean bands on the gel! I also tried the same protocol without Q solution and it didn’t work – just showing how important PCR additives can be.
Buoyed by success I tried a mixture of beetle families, and a again success. Then I tried COI Folmer primers with a lower annealing temperature- again success! Thormann et al’s protocol seems to be quite flexible and deals with pitfall trapped beetles of many family’s well.
Now we will see if it works for other markers…..
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…..
Really interesting article about male dung beetle competition. A really neat design with interesting results.
I’ve always thought that colour was going to be a useful functional trait for beetles. I’ve seen (or at least I think I’ve seen – I am colour blind!) a shift in colour of elytra/legs in my specimens from young forest to oldgrowth, but how to quantify this I had no idea until recently. Some other authors have used colour categories – but I think it could be a bit more subtle than that. Also, being heavily influenced by Violle’s definition of functional traits (http://onlinelibrary.wiley.com/doi/10.1111/j.0030-1299.2007.15559.x/full), I always prefer continuous quantitative traits.
So I went down the spectrometer path and was quickly overwhelmed by the options. After much research I’ve decides to go with the Ocean Optics USB 4000 http://www.oceanoptics.com/products/usb4000.asp. Possibly overkill – but it’s well recommended and I got a reasonable price in the end. I’m looking forward to testing it out in a couple of weeks!