November 20, 2019 - Oyster Species Determination using PCR
Today, I worked with Sam to create a master mix for PCR analysis of the samples that were received from the oyster distribution company. The samples that I had to create master mixes for are: 9, 10, 11, 12, 21, 22, 23, 24, 33, 34, and 35, with one extra tube being used as a negative control.
Using primer stocks with a concentration of 100 uM and a PCR reagent of 2x, I converted the following concentrations using the equation C1V1 = C2V2 to create a master mix containing a total of 25 uL per tube with the following concentrations:
| Materials | Volumes | Number of Reactions | Total Volume |
|---|---|---|---|
| DNA | 4 uL | N/A | N/A |
| COF | 0.15 uL | 18 | 2.7 uL |
| COR | 0.15 uL | 18 | 2.7 uL |
| COCqi | 0.1 uL | 18 | 1.8 uL |
| COCsi | 0.1 uL | 18 | 1.8 uL |
| PCR Reagent | 12.5 uL | 18 | 225 uL |
| Water | 8 uL | 18 | 144 uL |
| Total | 25 uL | — | add 21 uL of this total to each tube |
Once the appropriate master mix and DNA sample amounts were put into their respective tubes, they were put into a microcentrifuge to place all of the liquid in the tube to the bottom, and then all of the samples were placed into a thermal cycler to amplify the DNA in each of the samples using PCR. The cycling parameters for the PCR are 1 cycle at 95C for 10 minutes followed by 30 cycles at 95 for 1 minute, 51C for 1 minute, and 72C for 1 minute (reference is here).
Next Steps
The next step will be to insert the samples into a gel once the PCR is finished.