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