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|
|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).
The next step will be to insert the samples into a gel once the PCR is finished.