March
23
Lab 10
3/22/18
Today in lab we redid the PCR procedure with the cox1 and v4 primer. We then made our agarose gel.
Procedures
PCR
- Label 6 tubes with the numbers 1-6 on top. They all should already have the 12.5 ul of master mix in them
- In tube 1 add 0.6 uL of the Cox1 and 11.87 uL of water. This is the (-) control of the Cox1 Primer
- In tube 2, add 1 uL of the DNA, 0.63 uL of the Cox1 and 10.9 uL of water. This is the (+) control of the Cox1 Primer
- In tube 3, add 1 ul of control paramecium tube, 0.63 uL of the Cox1 and 10.9 uL of water. This is the eDNA of the Cox1 Primer
- In tube 4, add 0.63 uL of the V4 and 11.87 uL of water. This is the (-) control of the V4 Primer
- In tube 5, add 1 uL of the DNA, 0. 6 uL of the V4 and 10.9 uL of water. This is the (+) control of the V4 Primer
- In tube 6, add 1 ul of the control paramecium, 0.63 uL of the V4 and 10.9 uL of water. This is the eDNA of the V4 Primer
- Store the tubes in a rack at the front of the class and label where they are on the sheet of paper
Agarose Gel
- Pour 90 ml of DI water and 10 ml of TAE 10x into a flask
- In a different flask, obtain 35 ml of the TAE 10x mixture and combine with .6 g of agarose then mix
- Heat in a microwave for one minute and 20 seconds on power 70
- Cool the flask in a cooling bath for about 5 minutes.
- Set up your electrophoresis tray
- Pour the agarose gel solution into the tray and allow to cool
- Label the tray and then place in the designated area
Observations
| Cox1 Primer | 1 (-) control | 2 (+) control | 3 eDNA | V4 Primer | 4 (-) control | 5 (+) control | 6 eDNA | |
| 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 | 0.6 µL | 0.6 µL | 0.6 µL | 20 µM V4 | 0.6 µL | 0.6 µL | 0.6 µL | |
| Water | 11.9 µL | 10.9 µL | 10.9 µL | Water | 11.9 µL | 10.9 µL | 10.9 µL | |
| Total Volume | 25 µL | 25 µL | 25 µL | Total Volume | 25 µL | 25 µL | 25 µL |
Thoughts
We messed up, of course because what is learning without making mistakes? We put the cox 1 primer in all 6 tubes instead just the first 3 so we had to redo those 3 tubes. We labeled the tubes as with 1-6 on each of them with a SG on the top and a KSA on the side. Our tubes where in the second row from the bottom in the storage holder. Our Agarose Gel was labeled “Kaitlyn, Sydney, Angelo Group 8”.