3/22/2018
Today we performed polymerase chain reaction (PCR) on our environmental sample and our controls in order to prepare them for gel electrophoresis. We also made the agarose gel which we will be using to conduct the electrophoresis.
These are the mixtures we used for PCR
COX 1 V4
| 1 Negative Control | 2 Positive Control | 3 Experimental Sample | 4 Negative Control | 5 Positive Control | 6 Experimental Sample | |||
| 2X Master Mix | 12.5 µL | 12.5 µL | 12.5 µL | 2X Master Mix | 12.5 µL | 12.5 µL | 12.5 µL | |
| DNA | 0 µL | 1 µL | 1 µL | DNA | 0 µL | 1 µL | 1 µL | |
| 20 µM COX1 | 0.63 µL | 0.63 µL | 0.63 µL | 20 µM V4 | 0.63 µL | 0.63 µL | 0.63 µL | |
| Water | 11.87 µL | 10.87 µL | 10.87 µL | Water | 11.87 µL | 10.87 µL | 10.87 µL | |
| Total Volume | 25 µL | 25 µL | 25 µL | Total Volume | 25 µL | 25 µL | 25 µL |
PCR Procedure:
we prepare 6 small microfuge tubes, 3 with COX 1 primers and 3 with V4 primers. Each of the other 3 substances were pipetted into each tube following the volumes listed above. Each is then initialed and stored for later use.
Agarose Gell Procedure:
We first made a 100 mL solution of 1x TAE by mixing 10 mL of 10x TAE into 90 mL of D.I. water. Next we add 35 mL of this solution and 0.6 g of agarose to a separate beaker. A lid is placed loosly on the flask and the whole thing is microwaved for 80 seconds on power 7. The new solution is swirled and placed in a cooling bath for 5 minutes. Next, we set up a gel mold. Once the agarose solution had cooled we poured it into the mold and let it set.
The six indicated tubes are the ones my group completed
Pouring the agarose solution into the gel mold