Week 4 lab was an extension of the Sushi Lab started in Week 2.
- We removed our PCR tubes with our samples of fish tissue from ice and given time to thaw.
- We laid out 15 dots of about 1 microliter of loading dye on a sheet of parafilm.
- We recorded the names and order of samples on a piece of paper.
- Each lab member pipetted 3 microliters of each PCR product onto a single dot of loading dye, changing pipette tips in between each sample.
- We loaded each dot of loading dye mixed with PCR product into a cell in the gel setup, including a control in the first cell and a ladder in the 15th
- We fastened the lid to the gel and turned on the machine to run at 130 volts for 30 minutes.
Clean-Up of PCR products for sequencing – ExoSAP
- We determined the number of PCR clean-ups for our table, to prepare sufficient master mix. This included one for each sample, and a few extra, for a total of 18.
- The master mix recipe was prepared as follows:
Master Mix: Per Rxn Total Rxn: 18
H2O 10.59 uL _____190.6_______
10x buffer (Sap 10x) 1.25 uL _____22.5______
SAP 0.44 uL ______7.9________
Exo 0.22 uL ______4.0______
Master Mix Total 12.5 uL _______225_______
PCR Product 7.5 ul
Total Cleaned-up Volume 20.0 uL
3. We put each reagent in the ice bucket on the table.
4. We pipetted 7.5 uL of each PCR product into a clean, labeled 0.2 uL PCR tube.
5. We made the ExoSap master mix, keeping the reagents on ice while it was made.
6. We pipetted 12.5 uL into each PCR product tube.
7. We placed the tubes in a thermocycler and started the EXOSAP program.
8. After the program was done (~ 45 minutes later), Dr. Paul placed the PCR tubes in a labeled tube rack and placed them in the freezer.