IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/10

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8/10/10

9:30

PCR

Ingredient Amount Mastermix (4 reactions)
10X Buffer 2 μL 8 μL
10 mM dNTPs .4 μL 1.6 μL
Template .9 μL (18 ng) 3.6 μL
F Primer .4 μL added individually to each reaction
R Primer .4 μL added individually to each reaction
DMSO 1 μL/td> 4 μL
Polymerase .3 μL 1.2 μL
H2O 14.6 μL 58.4 μL

Per Reaction: 19.2 μL Mastermix, .4 μL F Primer, .4 μL R Primer.

Samples:

  • LamB F1R
  • LamB F2R
  • Negative control (no primer)

Primers:

  • LamBF1Xba
  • LamBF2Xba
  • LamBRSpe

Program:

95°C 3 min
95°C 30 sec
53°C 30 sec
72°C 2 min
go to step 2 31X
72°C 5 min
hold at 4°C

10:20 AM

PCR cleanup

Qiagen Kit

Concentrations:

  • pArsRF1R2 2' 28.8 ng/μL
  • pARsRF1R2 1 24 ng/μL
  • pArsRF1R2 2 30 ng/μL
  • pRpoS438 1 8 ng/μL
  • pRpoS438 2 7 ng/μL

11:00 AM

Site Directed Mutagenesis PCR

Ingredient Amount Mastermix (6 reactions)
10X Buffer 5 μL 30 μL
10 mM dNTPs 1 μL 6 μL
Template 20 ng added individually to each reaction
Primer 1 2 μL added individually to each reaction
Primer 2 2 μL added individually to each reaction
Polymerase 1 μL 6 μL
H2O 35 μL 210 μL

Samples:

  1. pArsRF1R2 2'
  2. pARsRF1R2 1
  3. pArsRF1R2 2
  4. pRpoS438 1
  5. pRpoS438 2

Program:

95°C 3 min
95°C 30 sec
64°C 1 min
72°C 1 min
go to step 2 12X
72°C 1 min
hold at 4°C

Primers:

10 micromolar

  • SDM2 pArsR RFC10 F502
  • SDM1 pArsR RFC10 F502
  • SDM2 pRpoS RFC10 F438
  • SDM2 pRpoS RFC10 F438

1:35 PM

Gel

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 LamBF1R 5 μL sample, 1 μL dye
3 LamBF2R 5 μL sample, 1 μL dye
4 LamBF2R 5 μL sample, 1 μL dye
5 negative control 5 μL sample, 1 μL dye

UIUC 8.10.10 lamB.jpg

PCR cleanup:

  1. pArsRF1R2 2'
  2. pARsRF1R2 1
  3. pArsRF1R2 2
  4. pRpoS438 1
  5. pRpoS438 2

all around 4 ng/μL

Dpn1 Digestion:

DNA 28 μL
H2O 15.5 μL
Buffer 4 5 μL
BSA .5 μL
DpnI 1 μL

incubate at 37°C for 1 hour, inactivate at 80°C for 20 min.

Samples:

  1. pArsRF1R2 2'
  2. pARsRF1R2 1
  3. pArsRF1R2 2
  4. pRpoS438 1
  5. pRpoS438 2