Date: September 17, 2019
Electrophoresis of PCR products
We first obtained our four PCR tubes and allowed them to thaw while we prepared the gel. We used a 10 µl micropipette to load sixteen 1 µl dye dots across a sheet of parafilm. Then, we pipetted 3-5 µl of PCR product into each dye dot. We used one additional dye dot as a negative control and two additional dye dots as a ladder. As we added our PCR product, we were recording the precise location of where each PCR sample went and who each sample belonged to. We set our micropipette to 5µl and used a filtered tip. We used the micropipette to suck up the dye dot and PCR mixture from the parafilm and transfer them into the wells of the gel. After all the wells were filled, the gel was run at 130 voltz for 30 minutes.
Clean-up of PCR products for sequencing – ExoSAP
I first obtained a strip of 8 new 0.2µl PCR tubes that I shared with my lab partner. We labeled each one with our individual sample codes without dividing the individual tubes. My sample codes were; EB01, EB02, EB03, EB04. After we labeled the tubes, we proceeded to make the ExoSAP Master Mix for our table. We decided to run 18 PCR clean-ups to account for pipette errors. We first had to multiply each ingredient in our master mix by 18 to get the correct measurements to begin making the master mix. Our masters mix consisted of; 190.6 µl of H2O, 22.5µl of 10x buffer (SAP 10x), 7.92µl of SAP, and 3.96µl of Exo. All of these reagents were kept on ice throughout the process of making the master mix. The total volume of the master mix was 225µl. We then added 7.5µl of each of our PCR products into the new clean PCR tubes we had just labeled. Next, we used a 20µl micropipette to place 12.5µl of the mix into each individual PCR tube. The tubes were all sealed with their caps and placed in the thermocycler to start the EXOSAP program. Professor Paul removed the PCR tubes after 45 minutes of running the program, and placed them in a labeled tube rack and placed in the freezer.
