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

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