IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/19

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8/19/10
10:00 AM

PCR

Samples:

  ArsB50  ArsB53  ArsB56  Negative Control (no primers) 

Primers:

  ArsBF2.0   ArsBR2.0 </li> </ul>

Program:

11:15 AM

Made glycerol stock of LamBF2

.9 ML 50% glycerol, .9 mL LamBF2 overnight.

11:20 AM

Digestion ArsRSDM2

B0015

pSB1T3

Incubate at 37 for 15°C min. Inactivate at 80°C for 20 min.

1:15 PM

Ligation

ArsRSDM2-B0015-pSB1T3

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

1:50 PM

Transformation

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

2:20 PM

Gel



It's hard to see, but there are very faint bands around 1.3 Kb which is the expected size for ArsB

Made 5 mL Cultures (2 colonies from each plate section)of the following samples:   pArsRF1R2 1</li>  pRpoS438 electroporated</li>  pRpoS438 heat shock</li>  ArsRSDM2-B0015-pSB1T3</li> </ul>

ArsRSDM2-B0015-pSB1T3 plate had questionable looking colonies (hence re-doing yesterday's procedure today) but I picked what looked like colonies anyways.

No growth on pArsRF1R2 2 or pArsRF1R2 2' plates.

3:15 PM

Plating

Plated 300 μL transformation product on Tet plate

3:30 PM

PCR Cleanup

ArsB50: 4.8 ng/μL ArsB53: 6.1 ng/μL ArsB56: 6.3 ng/μL

Francis Miniprepped ArsRSDM2 and LamBF2 overnights, both at 250 ng/μL.


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