Becker Lab qPCR troubleshooting update

Backstory

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.

Trap 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.

08012016_mocksample1_averagedbioreps

screen-shot-2016-08-01-at-3-06-29-pm

Experiment: 08012016_MockTrapSamples_DNAExtractionTest  Selected Filter: FAM (465-510)
Include Color Pos Name # larvae Cp Concentration Standard Average StDev
TRUE 65280 A4 Sample 4 M1Oly1SC 1 0 28.472 1.185609548
TRUE 65280 A5 Sample 4 M1Oly1SC 1 0
TRUE 65280 A6 Sample 4 M1Oly1SC 1 0
TRUE 255 A7 Sample 7 M2Oly1SC 1 29.07 1.52E-01 0
TRUE 255 A8 Sample 7 M2Oly1SC 1 28.72 1.82E-01 0
TRUE 255 A9 Sample 7 M2Oly1SC 1 26.59 5.50E-01 0
TRUE 255 A10 Sample 10 M3Oly1SC 1 29.74 1.07E-01 0
TRUE 65280 A11 Sample 10 M3Oly1SC 1 0
TRUE 255 A12 Sample 10 M3Oly1SC 1 28.24 2.34E-01 0
TRUE 255 B4 Sample 16 M1Oly5SC 5 19.75 1.91E+01 0 23.99833333 4.366469588
TRUE 255 B5 Sample 16 M1Oly5SC 5 19.75 1.91E+01 0
TRUE 255 B6 Sample 16 M1Oly5SC 5 20.7 1.17E+01 0
TRUE 65280 B7 Sample 19 M2Oly5SC 5 0
TRUE 65280 B8 Sample 19 M2Oly5SC 5 0
TRUE 65280 B9 Sample 19 M2Oly5SC 5 0
TRUE 255 B10 Sample 22 M3Oly5SC 5 26.84 4.82E-01 0
TRUE 255 B11 Sample 22 M3Oly5SC 5 28.79 1.76E-01 0
TRUE 255 B12 Sample 22 M3Oly5SC 5 28.16 2.43E-01 0
TRUE 255 C4 Sample 28 M1Oly10SC 10 27.55 3.34E-01 0 27.05444444 1.376218652
TRUE 255 C5 Sample 28 M1Oly10SC 10 27.96 2.70E-01 0
TRUE 255 C6 Sample 28 M1Oly10SC 10 27.75 3.01E-01 0
TRUE 255 C7 Sample 31 M2Oly10SC 10 25.16 1.15E+00 0
TRUE 255 C8 Sample 31 M2Oly10SC 10 25.31 1.07E+00 0
TRUE 255 C9 Sample 31 M2Oly10SC 10 25.35 1.05E+00 0
TRUE 255 C10 Sample 34 M3Oly10SC 10 28.67 1.87E-01 0
TRUE 255 C11 Sample 34 M3Oly10SC 10 27.62 3.22E-01 0
TRUE 255 C12 Sample 34 M3Oly10SC 10 28.12 2.48E-01 0
TRUE 65280 D4 Sample 40 M1Oly15SC 15 0 25.36333333 0.663073651
TRUE 65280 D5 Sample 40 M1Oly15SC 15 0
TRUE 65280 D6 Sample 40 M1Oly15SC 15 0
TRUE 255 D7 Sample 43 M2Oly15SC 15 25.66 8.90E-01 0
TRUE 255 D8 Sample 43 M2Oly15SC 15 24.41 1.70E+00 0
TRUE 255 D9 Sample 43 M2Oly15SC 15 25.35 1.05E+00 0
TRUE 255 D10 Sample 46 M3Oly15SC 15 26.32 6.31E-01 0
TRUE 255 D11 Sample 46 M3Oly15SC 15 24.87 1.34E+00 0
TRUE 255 D12 Sample 46 M3Oly15SC 15 25.57 9.32E-01 0
TRUE 255 E4 Sample 52 M1Oly20SC 20 23.56 2.65E+00 0 24.10555556 4.23596834
TRUE 255 E5 Sample 52 M1Oly20SC 20 22.99 3.56E+00 0
TRUE 255 E6 Sample 52 M1Oly20SC 20 22.53 4.53E+00 0
TRUE 255 E7 Sample 55 M2Oly20SC 20 20.48 1.31E+01 0
TRUE 255 E8 Sample 55 M2Oly20SC 20 17.88 5.05E+01 0
TRUE 255 E9 Sample 55 M2Oly20SC 20 21.63 7.22E+00 0
TRUE 255 E10 Sample 58 M3Oly20SC 20 29.57 1.17E-01 0
TRUE 255 E11 Sample 58 M3Oly20SC 20 28.59 1.95E-01 0
TRUE 255 E12 Sample 58 M3Oly20SC 20 29.72 1.09E-01 0
TRUE 65280 F4 Sample 64 M1Oly25SC 25 0 23.42333333 0.372066302
TRUE 65280 F5 Sample 64 M1Oly25SC 25 0
TRUE 65280 F6 Sample 64 M1Oly25SC 25 0
TRUE 65280 F7 Sample 67 M2Oly25SC 25 0
TRUE 65280 F8 Sample 67 M2Oly25SC 25 0
TRUE 65280 F9 Sample 67 M2Oly25SC 25 0
TRUE 255 F10 Sample 70 M3Oly25SC 25 23.83 2.30E+00 0
TRUE 255 F11 Sample 70 M3Oly25SC 25 23.34 2.96E+00 0
TRUE 255 F12 Sample 70 M3Oly25SC 25 23.1 3.37E+00 0

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.

 

 

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