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

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 </li> </ul>

Program:

10:20 AM

PCR cleanup

Qiagen Kit

Concentrations:

 pArsRF1R2 2' 28.8 ng/μL</li> pARsRF1R2 1 24 ng/μL</li> pArsRF1R2 2 30 ng/μL</li> pRpoS438 1 8 ng/μL</li> pRpoS438 2 7 ng/μL</li> </ul>

11:00 AM

Site Directed Mutagenesis PCR

Samples:

<ol>  pArsRF1R2 2' </li>  pARsRF1R2 1</li>  pArsRF1R2 2</li>  pRpoS438 1</li>  pRpoS438 2</li> </ol>

Program:

Primers:

10 micromolar

  SDM2 pArsR RFC10 F502</li>  SDM1 pArsR RFC10 F502</li>  SDM2 pRpoS RFC10 F438</li>  SDM2 pRpoS RFC10 F438</li> </ul>

1:35 PM

Gel



PCR cleanup: <ol> <li> pArsRF1R2 2' </li> <li> pARsRF1R2 1</li> <li> pArsRF1R2 2</li> <li> pRpoS438 1</li> <li> pRpoS438 2</li> </ol>

all around 4 ng/μL

Dpn1 Digestion:

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

Samples: <ol> <li> pArsRF1R2 2' </li> <li> pARsRF1R2 1</li> <li> pArsRF1R2 2</li> <li> pRpoS438 1</li> <li> pRpoS438 2</li> </ol>


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