Test PCRs with New 130kb Region PCR Primers

20220428 130-3 PCR

  • Using Primers 129778-F and 130860-R
  • PCR product should be 1,083bp
  • Made master mix on ice:
    • 11ul GoTaq
    • 0.55ul 129778 primer
    • 0.55ul 130860 primer
    • 7.7ul molecular grade water
  • Vortexed and spun down mix
  • Added 9ul of mix to strip tubes
  • Added 1ul 2mL sample DNA to the sample tube
  • Added 1ul molecular grade water to the negative tube
  • Vortexed and spun down tubes
  • Placed in the thermocycler DiNV1303 program:
    • 95 degrees C 2 minutes
    • 95 degrees C 30 seconds
    • 52 degrees C 30 seconds
    • 72 degrees C 1 minute
    • 72 degrees C 5 minutes
    • 12 degrees C hold
    • Italics lines are cycled 34 lines
  • Program rand for 1 hour and 52 minutes
  • Ran on a 1% gel for 35 minutes at 90 volts:

One band at the correct size, this is great!

20220409 130-4 PCR

  • Using Primers 129666-F and 131122-R
  • PCR product should be ~1.5bp
  • Made master mix on ice:
    • 11ul GoTaq
    • 0.55ul 129666 primer
    • 0.55ul 131122 primer
    • 7.7ul molecular grade water
  • Vortexed and spun down mix
  • Added 9ul of mix to strip tubes
  • Added 1ul 2mL sample DNA to the sample tube
  • Added 1ul molecular grade water to the negative tube
  • Vortexed and spun down tubes
  • Placed in the thermocycler DiNV1303 program:
    • 95 degrees C 2 minutes
    • 95 degrees C 30 seconds
    • 52 degrees C 30 seconds
    • 72 degrees C 1 minute 30 seconds
    • 72 degrees C 5 minutes
    • 12 degrees C hold
    • Italics lines are cycled 34 lines
  • Program rand for 1 hour and 52 minutes
  • Ran on a 1% gel for 35 minutes at 90 volts:

With no band, I decided to try a gradient PCR next.

20220504 130-4 Gradient

  • PCR didn’t work with 52 degrees, try a gradient under that
  • Set up gradient:
    • A: 52 deg
    • B: 51 deg
    • C: 49.5 deg
    • D: 47.4 deg
    • E: 44.6 deg
    • F: 42.2 deg
    • G: 40.8 deg
    • H: 40 deg
  • Not going to use row A because I already know it doesn’t work
  • Made master mix on ice:
    • 75ul GoTaq
    • 3.75ul 129666 primer
    • 3.75ul 131122 primer
    • 52.5ul molecular grade water
  • Vortexed and spun down mix
  • Aliquoted out 9ul mix into tubes, 2 per temp
  • Added 1ul 2mL sample DNA to the sample tubes
  • Added 1ul molecular grade water to the negative control samples
  • Ran PCR program same as above but with the gradient for the annealing temperature
  • Ran a 1% gel for 35 minutes at 90 volts: