IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/09/17

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9/17/10

8:15 AM

PCR

Ingredient Amount Mastermix (5 reactions)
10X Buffer 2 μL 10 μL
10 mM dNTPs .4 μL 2 μL
Template 1 μL (20 ng) 5 μL
F Primer .4 μL added individually to each reaction
R Primer .4 μL added individually to each reaction
Polymerase .2 μL 1 μL
H2O 15.6 μL 78 μL

Samples:

  1. pArsR
  2. pRpoS
  3. (+) control ArsR
  4. (-) control H2O

Primers (10 micromolar):

  • pArsR F1 2.0
  • pArsR R2 2.0
  • pRpoS F438 2.0
  • pRpoS R 2.0
  • ArsR RFC10 EcoRI
  • ArsR RFC10 EcoRI

Program:

95°C 3 min
95°C 30 sec
55°C 30 sec
72°C 1 min
go to step 2 30X
72°C 3 min
hold at 4°C

10:30 AM time

Gel

TAE, 1%, 175V


Lane Sample Amount
1 1 Kb Ladder 1 μL
2 pArsR 1 μL dye, 5 μL sample
3 pRpoS 1 μL dye, 5 μL sample
4 Positive control (ArsR) 1 μL dye, 5 μL sample
5 Negative control 1 μL dye, 5 μL sample


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