IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/10/03

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10.3.10

10:30

diluted minipreps from yesterday to 20 ng/μL for PCR

PCR

Ingredient Amount Mastermix (10 reactions)
10X Buffer 2 μL 20 μL
10 mM dNTPs .4 μL 4 μL
Template 20 ng (1 μL) added individually to each reaction
F Primer .4 μL 4 μL
R Primer .4 μL 4 μL
Polymerase .2 μL 2 μL
H2O 15.6 μL 156 μL

Samples

  1. J2334A215C 1
  2. J2334A215C 2
  3. J2334A215C 3
  4. J2334A215C 4
  5. ArsRSDM2C 1
  6. ArsRSDM2C 2
  7. ArsRSDM2C 3
  8. (+) control ArsR
  9. (-) control H2O

Primers

  • VF2
  • VR
  • ArsRF EcoRI
  • ArsR R

Program

95°C 3 min
95°C 30
55°C 30 sec
72°C 90 sec
go to step 2 30X
72°C 3 min
hold at 4°C

Gel: TAE 1% 175V

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 J2334A215 1 5 μL 1 μL dye
3 J2334A215 2 5 μL 1 μL dye
4 J2334A215 3 5 μL 1 μL dye
5 J2334A215 4 5 μL 1 μL dye
6 ArsRSDM2 1 5 μL 1 μL dye
7 ArsRSDM2 2 5 μL 1 μL dye
8 ArsRSDM2 3 5 μL 1 μL dye
9 (+) control ArsR 5 μL 1 μL dye
10 (-) control 5 μL 1 μL dye


UIUC 10.3.10.JPG