User:DrJones1935/17 July 2012

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I. Dilute primers to 10 uM working stock

  • Mix:
  • 2 uL 200 uM primer stock
  • 38 uL sterile, DNAse-, RNAse-free dH2O
  • Note: I think that the p5 primers got mixed up for forward and reverse, but it should not make a difference for the PCR

II. PCR

  • Mix Reagents:
  • Mix according to the following table, where 1 = pT5, 2 = LacZ, 3 = CAT*, 4 = tetr, 5 = T1, 0.1 = +template, and 0.2 = -template
  • For example, 1.1 = pT5 sample, 1.2 = pT5 control
1.1 1.2 2.1 2.2 3.1 3.2 4.1 4.2 5.1 5.2
Reagent (uL) (uL) (uL) (uL) (uL) (uL) (uL) (uL) (uL) (uL)
water 32.5 33.5 33.5 33.5 33.5 33.5 32.5 33.5 32.5 33.5
5x Phusion HiFi buffer 10 10 10 10 10 10 10 10 10 10
10 mM dNTP mix 1 1 1 1 1 1 1 1 1 1
10 uM primer (F) 2.5 (p1f) 2.5 (p1f) 2.5 (p2f) 2.5 (p2f) 2.5 (p3f) 2.5 (p3f) 2.5 (p4f) 2.5 (p4f) 2.5 (p5f) 2.5 (p5f)
10 uM primer (R) 2.5 (p1r) 2.5 (p1r) 2.5 (p2r) 2.5 (p2r) 2.5 (p3r) 2.5 (p3r) 2.5 (p4r) 2.5 (p4r) 2.5 (p5r) 2.5 (p5r)
Template 1 (pBAV1K-T5-gfp) 0 ECNR2 colony* 0 ECFI5 colony* 0 1 (pBR322) 0 1 (pBAV1K-T5-gfp) 0
Phusion HiFi polymerase 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

*For these templates, I touched a colony with a sterile pipette tip and spread it on the bottom of the PCR tube before adding reagents

  • Run PCR
Step Temp (oC) Time
1 94 4 m
2 94 30 s
3 53 30 s
4 72 1.5 m
5 GOTO 2 7x
6 94 30 s
7 66 30 s
8 72 1.5 m
9 GOTO 6 30x
10 72 5 m
11 4 ∞*

*After the cycle ended I moved the tubes to the deli refrigerator for the night

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