In today’s Lab 2 distinct PCR reactions were performed. One, labeled Test PCR, was used to create a library for M. Gattatus samples. The second PCR was to actually generate data on the M. Gattatus samples. For the final PCR a unique barcode was added to identify which individuals generate which sequences.
Test PCR:
- Create a master mix with the following per reaction:
- 0.8µL NEB One-Taq 2x Master Mix
- 0.4µL 10 µM F Primer (used PCR1_6)
- 0.4µL 10 µM R Primer (used PCR2_6)
- 6.2µL H20
- Label a set of tubes with appropriate labels and add 1.0µL of restriction product to each tube.
- Add 15µL of master mix to each tube and run PCR at 94˚C for 2 minutes, then 20 cycles of 94˚C for 30 seconds, 60˚C for 30 seconds, 68˚C for 45 seconds.
- Run the products of Test PCR on 1.5% Agarose Gel with a 100bp ladder at 130V for 40 minutes.
Final PCR:
- For each DNA library set up 8 PCR reactions in 50µL total volume.
- Create a master mix with the following per reaction:
- 0.31 µL Phusion DNA Pol
- 6.25 µL 5X Phusion HF Buffer
- 1.56µL F Primer (used PCR1_X) (10mM)
- 1.56µL R Primer (used PCR2_7) (10mM)
- 0.63µL DNTPs (10mM)
- 0.94µL DMSO
- 10.75µL Pure H2O
- Label a set of tubes with appropriate labels and add 3.0µL of restriction product to each tube.
- Run Final PCR at 98˚C for 30 seconds, then 10-20 cycles of 98˚C for 10 seconds, 65˚C for 30 seconds, 72˚C for 30 seconds and a final extension at 75˚C for 5 minutes.
- Run 2µL of product from Final PCR on 1% agarose gel with 100bp ladder.