IGEM:PennState/Labbook/NoahJohnson/2007-7-25

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July 25, 2007

PCR Protocol for Pfu Polymerase

  • PCR with Pfu and Taq polymerases
  • DNA Template: CRP*
  • Primers: 3410 & 3411
  • Set-up:
    • 5uL 10x PfuUltra HF reaction buffer
    • 0.4uL dNTPs (25mM each dNTP)
    • 1uL DNA template (100ng/uL)
    • 1uL Primer #1 (3410)
    • 1uL Primer #2 (3411)
    • 1uL PfuUltra HF DNA polymerase (2.5 U/uL)
    • 40.6uL dH2O (Enough to make 50uL total)
  • Ran: (all at 72C)
    • 6 Pfu trials
    • 1 Taq trial
    • 1 negative

Thermocycler Protocol (pfu works):

  • Cycle 1: (1x)
    • Step 1: 95C for 1:00
  • Cycle 2: (28x)
    • Step 1: 95C for 0:15
    • Step 2: 54C for 0:30
    • Step 3: 72C for 2:00
  • Cycle 3: (1x) Hold at 4C


Ran Gel on PCR Products (Start @ 3:08 pm)

  • Samples:
    • 3uL PCR product
    • 1uL 5x buffer
    • 1uL Sybr Green
  • Ladder:
    • 1uL 1kb Ladder
    • 1uL 5x buffer
    • 1uL Sybr Green
    • 2uL TAE Buffer

Well#

  • 1
  • 2 I741023 Pfu 72C #1
  • 3 I741023 Pfu 72C #2
  • 4 I741023 Taq 72
  • 5 (-) Pfu 72C
  • Helped make RP3087 competent cells
  • Plated motility parts and left in incubator overnight

9:30-6

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