This week we ran our DNA samples from the Sushi Test on gel electrophoresis and ‘cleaned up’ the PCR reaction using an ExoSap master mix. To begin, I took my samples from the ice that they were previously stored in and thawed it at room temperature. While they were thawing, we (Mikayla) dotted out 19 loading dye dots on a sheet of parafilm- each dot was about one microliter. The protocol said to put 16 dots but since we had a negative control and a ladder on each row we had three more. After prepping the parafilm, each person at my lab bench pipetted three microliters of their PCR product onto each dot, and Prof Paul put in the ladders on the parafilm for us before running the gel. I then pipetted all of our samples into the gel lanes. Tips were switched on the pipet between samples every time to avoid contamination. [The lanes and sample ID’s are shown below]. Finally, we ran the gel at 130V for 30 minutes.
|LANE # (L à R)||SAMPLE ID #|
The next step was to clean up our PCR products for future sequencing. At each table two partners shared a row of 8 0.2 microliter PCR tubes (4 each). We label them with our sample codes on the top, front and back to ensure that our sample would not be lost. Next, we made the master mix. Since we had 16 samples of fish DNA we made enough master mix for 18 reactions. [Recipe is shown below]. All the reagents were put on ice and we only took them off ice when we needed to use them. We pipetted 7.5 microliters of each of our PCR products into our new PCR tubes and then pipetted 12.5 microliter of the Master Mix we made into each of our little tubes. We them put them in the thermocycler and started the EXOSAP program.
(Prof. Paul did the last step: After 45 minutes of the program, PCR tubes were placed in a labeled tube rack and placed in the freezer)
|Master Mix||RXN (1)||RXNs: 18|
|H2O||10.59 microliters||190.6 microliters|
|10x buffer (Sap 10x)||1.25 microliters||22.5 microliters|
|SAP||0.44 microliters||7.92 microliters|
|Exo||0.22 microliters||3.96 microliters|
|Master Mix Total||12.5 microliters||225 microliters|