interesting Laby PCR

Sorry everyone. I meant to past this about two days ago and just now got a chance to do so. R: Below is a gel of PCR (with Laby WC primers) on the serial dilution of Laby + SW as well as the water samples at T0 and T24 (first replicate of each treatment only). The top row of 0-10 is DNA extraction of serial dilution via boil prep. The bottom row of 0-10 is DNA extraction of serial dilution via DNeasy kit.

D: Both DNA extraction methods work. However, Lisa told me today that we got more DNeasy kits in, so the boil method won’t be needed. I think it is nice to know that the boil method works on Laby for future reference. It looks like the limit of Laby detection by cPCR is 10^3 cells per mL, because there are no bands for either DNA extraction method from 10^-2 and lower. I think that this is weird and that we should have seen bands further along the dilution series. One possible explanation is that clumps of Laby in the undiluted were not broken up so that no cells (or very few) actually made it into the next tube.  The really interesting results are not in the dilution series but in the actual water samples collected. For T0, we only got a PCR band in treatments were we added Laby (SL and SOL). Sweet. Even more interesting, at T24, Laby was detected in the beakers without oysters but Laby was NOT detected it the beaker with the oysters. This pattern is what we would expect if the oysters really are filtering out the Laby. However, an alternative explanation is that by this time the Laby have all stuck to a surface of some kind and were not picked up when we sampled the water from the beaker. The second explanation is prob more likely because the eelgrass in all the beakers looked funky, despite the oysters. We will have a better understanding af what is going on in the eelgrass after histology and image analysis.


SL = DNA ladder       0 = 10^0 (aka undiluted) SW + Laby    1 = 10^-1 SW + Laby                       2 = 10^-2 SW +Laby      3 = 10^-3 SW +Laby                       4 = 10^-4 SW + Laby      5 = 10^-5 SW + Laby                      6 = 10^-6 SW +Laby        7 = 10^-7 SW + Laby                      8 = 10^-8 SW + Laby      9 = 10^-9 SW + Laby                    10 = 10^-10 SW + Laby S = SW only                                          SO = SW plus Oyster    SL = SW plus Laby          SOL = SW plus Oyster plus Laby T0 = 0 hr collection                          T24 = 24 hr collection (+) = Laby DNA                                                         (-) = NTC

I should have just done this today. Anyway, David still was unagreeable so I continued this data mining process by downloading Stephen’s output file. The rest is below.

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