IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/26

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8/26/10

9:00 AM

Dilutions to 20 ng/ul

Sample concentration Amount sample Amount water
pArsRF1R2SDM2 1 339.3 ng/μL 1.18 μL 18.82 μL
pArsRF1R2SDM2 2 344.2 ng/μL 1.16 μL 18.84 μL
pRpoS438SDM2 2 362.9 ng/μL 1.10 μL 18.90 μL

9:35 AM

Verification PCR

Ingredient Amount Mastermix (5 reactions)
10X buffer 2 μL 10 μL
10 mM dNTP .4 μL 2 μL
Template 1 μL (20 ng) added individually to each reaction
F primer .4 μL 2 μL
R Primer .4 μL 2 μL
Polymerase .3 μL 1.5 μL
H2O 15.5 μL 77.5 μL

Per Reaction: 19 μL mastermix, 1 μL template

Samples:

  • pArsRF1R2SDM2 1
  • pArsRF1R2SDM2 2
  • pRpoS438SDM2 2
  • negative control (no template)

Primers:

VF2, VR at 10 micromolar

Program:

95°C 3 min
95°C 30 sec
55°C 30 sec
72°C 45 sec
go to step 2 30X
72°C 2 min
hold at 4°C

12:50 PM

Digestion

B0015:

DNA 1.7 μL (~340ng)
H20 40.8 μL
Buffer4 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL

ArsRSDM2

DNA 17 μL (~340ng)
H20 25.5 μL
Buffer4 5 μL
BSA .5 μL
XbaI 1 μL
PstI 1 μL

pSB1T3

DNA .71 μL (~340 ng)
H20 41.79 μL
Buffer4 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL

Incubate at 37°C for 15 min, inactivate at 80°C for 20 min.

1:00 PM

PCR ArsB

Ingerdient Amount Mastermix (6 reactions)
10X Buffer 2 μL 12 μL
10 mM dNTPs .4 μL 2.4 μL
Template .9 μL (18 ng) added individually to each reaction
F Primer .4 μL 2.4 μL
R Primer .4 μL 2.4 μL
Polymerase .3 μL 1.8 μL
H2O 14.6 μL 87.6 μL
DMSO 1 μL 6 μL

Samples:

  • ArsB 1
  • ArsB 2
  • ArsB 3
  • ArsB 4
  • negative control (no template)

Primers:

ArsB F 2.0, ArsB R 2.0 at 10 micromolar

Program:

95°C 3 min
95°C 30 sec
53°C 30 sec
72°C 2 min
go to step 2 32X
72°C 5 min
hold at 4°C

2:00 PM

Gel

Lane Sample Amount
1 1 Kb ladder 1 μL
2 pArsRF1R2SDM2 1 5 μL sample, 1 μL dye
3 pArsRF1R2SDM2 2 5 μL sample, 1 μL dye
4 pRpoS438SDM2 2 5 μL sample, 1 μL dye
5 negative control 5 μL sample, 1 μL dye

Ligation

ArsRSDM2 2 μL
B0015 2 μL
pSB1T3 2 μL
H2O 11 μL
Ligation Buffer 2 μL
Ligase 1 μL

incubate at room temperature for 10 min, inactivate at 80°C for 20 min.

2:35 PM

Transformation

50 μL cells, 2 μL ligation product. 10 min on ice, 45 sec at 42°C, 2 min on ice. add 1 mL LB, recover for 1 hour at 37°C shaking.

3:10 PM

PCR Cleanup Qiagen kit

pRpoS438SDM2 2: 21.6 ng/μL pArsRF1R2SDM2 1: 21ng/μL pArsRF1R2SDM2 2: 22.7 ng/μL

3:45 PM

Sequencing reactions

  • 20-30 ng DNA
  • total volume 10 μL
  • 20 pmol primer
  • 1.5 μL DNA
  • 2 μL Primer (VF2)
  • 6.5 μL H2O
  • Samples:

    • pRpoS438SDM2
    • pArsRF1R2SDM2 1
    • pArsRF1R2SDM2 2

    4:00 PM

    plated 200 μL transformation on tet plate




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