The Becker lab is currently attempting to use qPCR to quantify Olympia oyster larvae in two sets of environmental samples. One set is from Fidalgo Bay 2013 research where the samples were collected using passive traps and are fairly sediment rich. The second set is from Megan’s samples where were collected by pumping and were taken in Summer 2015 in Dyes Inlet and Fidalgo Bay. To reference each of these studies I will call the first set Trap samples and the second set Pump samples, referencing the method of sample collection which is the major distinguishing factor between the samples.
Overview- We have been using a modified ProK method for DNA extraction of the trap samples however there have been questions on whether or not the samples may still even contain enough genetic material to run qPCR. In a previous discussion with the Roberts Lab I indicated that we have been doing a series of PCR experiments to test the presence of DNA using eukaryotic and bacteria primers. We were able to see a faint band in a subset of trap samples that were previously extracted using the modified ProK method, so we concluded that there is still DNA in the samples but I’d like to run a few more tests to completely confirm this.
In another discussion with the Roberts Lab I stated that we did use the modified Prok method on a spiked set of environmental samples and qPCR was successful with this experiment. I didn’t account for the fact that this experiment was run using plankton samples, pump samples not trap samples, however, and decided that we need to determine the usefulness of applying the ProK method to trap samples, which are more sediment rich than plankton samples. To do this we ran a series of experiments were three “bio reps” of a standard curve (1,5,10,15,20,25 spiked Oly larvae) were prepared using a previously collected trap sample. We pipetted out approximately 1ml of trap sample and placed into labeled 50ml tubes. To these tubes we spiked cultured Oly larvae that were previously fixed with DMSO and transferred into 95% ethanol to make 3 replicate standard curves spiked into sediment. These “bio reps” were then transferred to 2ml eppendorf tubes and spun down to pipette out the ethanol supernatant and set out to dry. When the samples were dry, we added 1ml of the ProK solution and incubated the samples at 56C for 23 hours. For the last hour of cooking, we incubated at 95C to denature ProK and then the samples were placed in the freezer until ready to use for qPCR. On 8/1/2016 and the results of this experiment are below.
|Experiment: 08012016_MockTrapSamples_DNAExtractionTest Selected Filter: FAM (465-510)|
Between the three BioReps (run in triplicate) there was a lot of variation using the ProK method of extraction and a number of questions arose.