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Lab 12:

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:

  1. Create a master mix with the following per reaction:
    1. 0.8µL NEB One-Taq 2x Master Mix
    2. 0.4µL 10 µM F Primer (used PCR1_6)
    3. 0.4µL 10 µM R Primer (used PCR2_6)
    4. 6.2µL H20
  2. Label a set of tubes with appropriate labels and add 1.0µL of restriction product to each tube.
  3. 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.
  4. Run the products of Test PCR on 1.5% Agarose Gel with a 100bp ladder at 130V for 40 minutes.

Final PCR:

  1. For each DNA library set up 8 PCR reactions in 50µL total volume.
  2. Create a master mix with the following per reaction:
    1. 0.31 µL Phusion DNA Pol
    2. 6.25 µL 5X Phusion HF Buffer
    3. 1.56µL F Primer (used PCR1_X) (10mM)
    4. 1.56µL R Primer (used PCR2_7) (10mM)
    5. 0.63µL DNTPs (10mM)
    6. 0.94µL DMSO
    7. 10.75µL Pure H2O
  3. Label a set of tubes with appropriate labels and add 3.0µL of restriction product to each tube.
  4. 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.
  5. Run 2µL of product from Final PCR on 1% agarose gel with 100bp ladder.

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