Professor John Paul
Lab 3 Entry
The Sushi Test (continued)
On September 12th, I returned to the lab and retrieved my PCR tubes that I put in the thermocycler on September 5th. Our group then brought our PCR tubes to the side benches in the lab and began setting up a new gel in order to do gel electrophoresis. One of my members (Kayla) used a micropipette and made four rows of three of 2 microliters of loading dye onto a small piece of parafilm. Kayla then added 3 microliters of each person’s PCR tubes in the correct corresponding rows of the droplets of loading dye, making sure to use a new pipette tip each time. I then used a microliter to transfer the 5 microliter samples from the parafilm into the wells of the gel, making sure to use different pipette tips each time and that the wells were labeled on a piece of separate paper and each person’s samples could be identified. Gel electrophoresis was then conducted for about 15 minutes.
Once approximately 15 minutes passed, our group collected the gel and took it to be observed under the machine for special imaging. The image showed that one of my group members had all three of their samples, two of my three samples worked (CP02 and CP03), and two of my members, unfortunately, had none of their samples work. My wells were the three on the far right on the top row.
Since two of my three samples worked (CP02 and CP03), I then continued with the ExoSap PCR Clean-Up protocol. I collaborated with a group of fellow classmates whose samples also worked. A master mix of containing water, 10x buffer, SAP, and Exo was made for 20 reactions and its combined total measured 250 microliters (12.5 microliters per reaction) and placed in ice. A long series of new PCR product tubes were provided by the professor and each of us received a certain number of these tubes (since I had two samples that worked, I got two tubes; my tubes were #10 for CP02 and #11 for CP03). I labeled and recorded my tubes with the appropriate identifications and proceded to transfer 7.5 microliters of my CP02 into #10 and 7.5 microliters of my CP03 into #11. Then I transferred 12.5 microliters of the ExoSap master mix into #10 and another 12.5 microliters into #11 (all while using clean pipette tips in between each transfer of liquids). Once each person finished establishing their PCR tubes, we all put our tubes together in a thermocycler. The lab period then ended and we were excused to leave. Our professor then put the tubes into a labeled tube rack and placed into the freezer.
On September 26th, we will continue with this lab and start the EXOSAP program.