Today we ran gels for our DNA extractions from last week and then set up and ran PCR.
As usual, the gels were already set up, so we mixed our extractions with loading dye on parafilm, then loaded the gel. We ran the gel at 140V for 20min, then imaged it (see below). All extractions were successful for the possums.
Next, we aliquoted 20 µL of each of our extractions into two more tubes to share with the other tables. Each table worked with a specific EPIC marker for all samples, so each table needed DNA extraction from each sample. We did however remove two samples in which the extraction did not work and two from a population that was overrepresented, so we were working with a total of 32 samples. The 32 samples were split among the 4 people at each table, so each person worked with 8 samples.
The three EPIC markers were 5334, 5525, and 5536. My table worked with 5536.
The samples that I prepped for PCR were:
|Tube Label||Specimen ID|
|1 AC||JP 1229|
|2 AC||JP 1299|
|3 AC||JP 1302|
|4 AC||JP 1309|
|5 AC||JP 1311|
|6 AC||JP 1315|
|7 AC||JP 1316|
|8 AC||JP 1322A|
We did the following steps to prep for PCR:
- Add 1 µL of each template DNA to appropriately labeled PCR tube; store on ice.
- Prep master mix for the table, keeping mix and reagents on ice. Angela did this for our table. Since we had 32 samples total, she prepped enough mix for 40 samples. Angela prepped all of the Master Mix in the order shown in the table below except for the taq, which JP added for everyone at the same time so that no one’s PCR tubes sat for too long before starting.
Material per rxn (µL) per 40 rxn (µL) ddH2O 15.0 600 10x buffer + Mg 2.00 80 BSA 1.00 40 dNTPs .20 8 F-primer 5536 .20 8 R-primer 5536 .20 8 Taq .04 1.6
- Once Master Mix was complete, I vortexed it briefly. Then I added 19 µL of the master mix to my PCR tubes that already had template DNA. I pipetted up and down to mix, then stored the samples on ice until PCR.