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

9:35 AM

Verification PCR

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:

12:50 PM

Digestion

B0015:

ArsRSDM2

pSB1T3

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

1:00 PM

PCR ArsB

Samples:

 ArsB 1  ArsB 2 </li> ArsB 3 </li> ArsB 4 </li>  negative control (no template)</li> </ul>

Primers:

ArsB F 2.0, ArsB R 2.0 at 10 micromolar

Program:

2:00 PM

Gel

Ligation

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</li> total volume 10 μL</li>  20 pmol primer</li> </ul>

  1.5 μL DNA </li>  2 μL Primer (VF2) </li>  6.5 μL H2O </li>

Samples:  pRpoS438SDM2</li> pArsRF1R2SDM2 1</li> pArsRF1R2SDM2 2</li> </ul>

4:00 PM

plated 200 μL transformation on tet plate


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