For the Pyrimidudes, Lab 4 was all about separating out and then cleaning up the products of our PCR reactions from the last lab section. In order to set up the gel, the Pyrimidudes planned out which samples would go where; in total, we had 13 samples, plus a ladder and control. In order to prep out PCR, products, I pipetted out 15 1.5 microliter dots of loading dye, while the rest of the Pyrimidudes carefully set up the PCR reactions for adding to the dots. Each of our group added 3 microliters of DNA to each dye dot, with the exception of myself, who added the dye dot and PCR products directly into each lane, where they were mixed in-situ by pipetting up and down. We then run the gel at 130 volts for 30 minutes.

As this was running, we next prepped at PCR cleanup using ExoSAP of some of the remaining samples by creating a master mix using 190.62 μL H2O, 22.5 μL 10x SAP Buffer, 7.9 μL EXP, and 3.96 μL SAP for a total volume of 225 μL. We then added 12.5 μL of this master mix to each of 13 different labeled PCR tubes, to which was added 7.5 μL each of our PCR products. The PCR products were then run for 45 minutes by Dr. Paul using the ExoSAP protocol.

As part of the field trip, I got to see and experience what the reality of what population genetics applies to in the real world. Going to all of the various field sites for the Mimulus guttatus populations, it was very obvious to me how these very different sites could favor a large variety of different alleles across the whole population, and how these populations could face a myriad of dispersal and survival challenges throughout their lives that impact the fixation of new alleles and the removal of old ones. The fact that such water-dependent flowers and survive in such a harsh environment as th serpentine barrens was very interesting to me!

For the DNA extraction procedure, I used 2 copies of one sample of fish labelled “Red Drum” that I purchased at the 88 Manor Market located near my apartment in San Leandro, CA. Tissue from the region near the gills was collected in 2 vials and stored on ice until the procedure. The samples were stored on ice in my freezer until the day of the labs, when they were needed. From the defrosted samples, I removed roughly 20 mg of tissue for each on the 2 copies and added them into a 1.5 mL reaction tube labelled with their designations (JK01 and JK02, respectively). To each tube, 100 microliters of extraction solution was added with a 200-microliter pipette, followed immediately by 25 microliters of tissue preparation solution.  The solutions were mixed by briefly vortexing. After this, the samples were mashed up with a pipette tip until thoroughly masticated and incubated at room temp for 10 minutes. After this, I moved the samples to the heat block and incubated for a further 3 minutes at 95 C. After this, 100 microliters of neutralizing solution was added to the reaction to stop it, and it was placed on ice until needed for PCR.

 

After samples were prepared, a PCR reaction was prepared to amplify ambient sample DNA for identification of species. To begin, a 1:10 dilution was prepared by pipetting 18 microliters of DI H2O into a 1.5 mL tube labelled “1:10 JK1” and “1:10 JK2”. To this, 2 microliters of the DNA extraction performed above was added. After this, the tube was vortexed in a centrifuge to consolidate all liquid and mix the tube. After this, a master mix reaction was created as per the protocol in the handout, with enough made for 16 reactions based on the 13 samples in our group (102.4 microliters water, 160 microliters rx mix, 12.8 microliters each of forward and reverse primer extract). However, we were short 4 reaction volumes, and another member of our group had to use a different PCR master mix. I took 2 18 microliter aliquots of this master mix and added them to 2 separate PCR tubes. To these tubes, I added one 2 microliter aliquot of DNA extraction each. After the thermocycler was set up, I added my tubes, after which they were PCRed at the following parameters:

 

4 min of 94 initial denaturation

30 cycles of:

94 C for 30 sec (denaturing)

52 C for 40 sec (annealing)

72 C for 1 min (extension)

72 C for 10 min (final extension)

10C for hold

 

After this, professor John R. Paul collected the samples and ran aliquots of the DNA to enable a positive genetic ID for each fish species present in the sample.

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