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.

Written on November 20, 2019