In the second part of of the DNA extraction lab from animal tissue derived from sushi samples, our lab group ran a gel electrophoresis and PCR clean up. The first step in this process was obtaining our saved PCR tubes and letting them thaw at room temperature, and then the samples were placed on ice. In order to use our samples in gel electrophoresis, we pipetted 16 dots of dye on a sheet of parafilm. Each dot was approximately 1.5 microliters. We then pipetted 3 microliters of each PCR product into its own dot while changing pipette tips in between each dot. Each member was responsible for their own PCR products. After each dot of dye had PCR product in it, we set a pipette to 5 microliters and loaded each dot into our gel. We ran the gel at 130 volts for 30 minutes.
While our samples were running gel electrophoresis, we started our PCR cleanup. We began by carefully labeling new 0.2 microliter PCR tubes with our sample codes. I labeled my tubes with GS01, GS02, Gs03, and GS04. We proceeded to make an ExoSap Master mix by mixing 211.8 microliters of pure water, 25 microliters of 10x buffer (Sap 10x), 88 microliters of SAP, and 4.4 microliters of Exo. We were able to do this successfully eventually, but initially, we did not use the correct amount of buffer or SAP, thus we had to redo our master mix. We mixed our successful master mix by holding the tube of master mix and waving our arm left and right on a horizontal plane, multiple times.
We pipetted 7.5 microliters of each PCR product into a clean, labeled, 0.2 microliter PCR tube. We also added 12.5 microliters of the master mix into the labeled PCR tube and then placed all PCR tubes into a thermocycler and started the EXOSAP program for approximately 45 minutes. When the program was completed, our professor placed the PCR tubes in a labeled rack and then they were placed in the freezer.