50ul PCRs for 77kb-DiNV To Make Enough For Cas9 Digestion Assay

20220414 77-DiNV-1

  • Basically I wanted to scale up my usual PCR ratio by 5
  • Going to use my 2mL sample as the sample and water as a negative control
  • Made master mix on ice:
    • 5ul GoTaq * 5 * 2.2 = 55ul
    • 0.25ul 77-F-1 * 5 * 2.2 = 2.75ul
    • 0.25ul 77-R-1 * 5 * 2.2 = 2.75ul
    • 3.5 ul H2O * 5 * 2.2 = 38.5ul
  • Vortexed and spun down mix
  • Added 45ul of mix to 2 PCR tubes
  • Added 5ul of 2mL DNA to the first tube
  • Added 5ul molecular grade water to the second tube
  • Vortexed and spun down PCR tubes
  • Placed PCR tubes in the thermocycler 77-DiNV-1 program
    • 95 degrees C 2 min
    • 95 degrees C 30 sec
    • 52 degrees C 1 minute
    • 72 degrees C 1 minutes, 30 seconds
    • 72 degrees C 5 min
    • 12 degrees C hold
    • Italic lines are cycled 34 times
  • I made sure to change the volume to 50ul
  • Afterwards 5ul of the PCR was run on a 1% gel for 35 minutes at 90V:

Looks good!

20220508 77-DiNV-2

  • I modified the PCR a little bit for this to get only 1 band (and at the right size)
  • I used 3/4th of the usual amount of primers
  • Made master mix on ice:
    • 5ul GoTaq * 5 * 2.2 = 55ul
    • 0.187ul 77-F-1 * 5 * 2.2 = 2.057ul
    • 0.187ul 77-R-1 * 5 * 2.2 = 2.057ul
    • 3.63 ul H2O * 5 * 2.2 = 39.93ul
  • Vortexed and spun down mix
  • Added 45ul of mix to 2 PCR tubes
  • Added 5ul of 2mL DNA to the first tube
  • Added 5ul molecular grade water to the second tube
  • Vortexed and spun down PCR tubes
  • Placed PCR tubes in the thermocycler 77-DiNV-1 program
    • 95 degrees C 2 min
    • 95 degrees C 30 sec
    • 53 degrees C 1 minute
    • 72 degrees C 2 minutes
    • 72 degrees C 5 min
    • 12 degrees C hold
    • Italic lines are cycled 34 times
  • I made sure to change the volume to 50ul
  • Afterwards 5ul of the PCR was run on a 1% gel for 35 minutes at 90V: