November
28
Lab 13: PCR & Posters
November 15, 2916
In this lab, we planned out the lay out of our poster presentation which includes the material that we have learned during this lab. The purpose of this lab was to prepare our DNA for PCR.
Procedure:
- Using the concentration of the DNA we had extracted from the week before, we calculated the volume of DNA necessary for today’s lab which was 3 microliters
- Calculate the volume that equals 1—ng
- 25 microliters of the correct primers in the tube
- (1.25 microliters)(20microMoles)= X(25 microliters)
- Final Concentration: 1 microMolar of each primer
- Calculate the volume of water to make the total solution volume 25 microliters
- For the PCR Assay, 25 microliters of reaction mixture is needed
- 5 microliters of AmpliTaq gold 360 2x Master Mix
- DNA depending on concentration which is calculated above
- For both sets of reagents, 1 control tube will need to be prepared but will contain water instead of DNA
- Mix samples by gently vortexing tubes to make sure everything is combined
- Thermal cycling is as follows:
- Initial Denaturation: 94 degrees Celsius for 2.5 minutes
- Denaturation 95 degrees Celsius for 30 seconds
- Primer Annealing: 58 degrees Celsius for 1 minute
- Primer elongation: 65 degrees for 2.5 minutes
- Extension – 72 degrees Celsius for 8 minutes
- Repeat steps b-e for 35 cycles
| Tube | Euk | Cox1 | Control Euk | Control Cox 1 |
| 2xTaq mix (mL) | 12.5 | 12.5 | 12.5 | 12.5 |
| DNA (mL) | 3 | 3 | 0 | 0 |
| 20 mL Euk primers (mL) | 1.25 | 0 | 1.25 | 0 |
| 20 mL COX1 Primers (mL) | 0 | 1.25 | 0 | 1.25 |
| Water (mL) | 8.25 | 8.25 | 11.25 | 11.25 |
| Total Volume (mL) | 25 | 25 | 25 | 25 |
Results/Conclusion:
The DNA extracted from the previous lab had a purity of 1.81. The tubes are ready for the gel electrophoresis! I am excited to run the gels to see what the bands will look like!.