Testing p47_q, 115, Lef 9 and Lef 4 PCR Primers on 3mLC Gel Extracted DNA. These Are Other Primers for DiNV

Lef_9 and Lef_4 PCRs 20220119

  • These two primers have similar sizes and annealing temps so I am going to do them at the same time
  • Tm is ~60 degrees C, so annealing temp I will try is 55 degrees C
  • Lef_9 is 941bp long and Lef_4 is 788bp long
  • Generated 10uM working stock primers from these
    • Each tube gets 90ul of molecular grade water
    • Then each tube gets 10ul of their respective F or R primer
  • Using GoTaq mix
  • There are 4 samples, and I need a neg and positive control too, so 6 samples
  • Made master mix for Lef_9:
    • 33ul GoTaq mix
    • 23.1ul molecular grade water
    • 1.65ul Lef_9_F primer
    • 1.65ul Lef_9_R primer
  • Made master mix for Lef_4:
    • 33ul GoTaq mix
    • 23.1ul molecular grade water
    • 1.65ul Lef_4_F primer
    • 1.65ul Lef_4_R primer
  • One set of strip tubes got 9ul of Lef_9 mix, one set of strip tubes got 9ul of Lef_4 mix
  • Each set got 1ul of DNA, or 1ul of H20 (neg control):
    • 1: 3mLC 150
    • 2: 3mLC UP
    • 3: 3mLC LOW
    • 4: 3mLC ADD
    • 5: Neg control
    • 6: Pos control
  • Samples were vortexed and spun down, then put into the PCR machine 55TEST long program:
    • 95 degrees C 3 minutes
    • 95 degrees C 30 seconds
    • 55 degrees C 30 minute
    • 72 degrees C 1 minute
    • 72 degrees C 5 minutes
    • 12 degree C hold
    • bold sections are cycled 34 times
  • Samples were put in the fridge afterward and gelled the next day
  • 20220120 1% gel ran at 90V for 35 min:

p47_q and 115 PCRs 20220120

  • These two primers have similar sizes and annealing temps so I am going to do them at the same time
  • Tm is ~60 degrees C, so annealing temp I will try is 55 degrees C
  • p47_q is 87bp in size and 115 is 43bp in size, very small!
  • Generated 10uM working stock primers for 115 (already had one for p47_q)
    • Each tube gets 90ul of molecular grade water
    • Then each tube gets 10ul of either 115_R or 115_R
  • Using GoTaq mix
  • There are 4 samples, and I need a neg and positive control too, so 6 samples
  • Made master mix for p47_q:
    • 33ul GoTaq mix
    • 23.1ul molecular grade water
    • 1.65ul p47_q_F primer
    • 1.65ul p47_q_R primer
  • Made master mix for 115:
    • 33ul GoTaq mix
    • 23.1ul molecular grade water
    • 1.65ul 115_F primer
    • 1.65ul 115_R primer
  • One set of strip tubes got 9ul of p47_q mix, one set of strip tubes got 9ul of 115 mix
  • Each set got 1ul of DNA, or 1ul of H20 (neg control):
    • 1: 3mLC 150
    • 2: 3mLC UP
    • 3: 3mLC LOW
    • 4: 3mLC ADD
    • 5: Neg control
    • 6: Pos control
  • Samples were vortexed and spun down, then put into the PCR machine 55TEST program:
    • 95 degrees C 3 minutes
    • 95 degrees C 30 seconds
    • 55 degrees C 30 minute
    • 72 degrees C 30 seconds
    • 72 degrees C 5 minutes
    • 12 degree C hold
    • bold sections are cycled 34 times
  • 20220120 2% gel ran at 90V for 30 min: