IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/03

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8/3/10
9:45 AM

Digestions

ArsR-B0015

B0034

pSB1C3

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

10:45 AM

Miniprep

Promega Kit

Concentrations:

pRpoS438 colony 1: 214.4 ng/μL

pRpoS438 colony 2: 380.3 ng/μL

pArsRF1R2 colony 1: 246.9 ng/μL

pArsRF1R2 colony 2: 456.2 ng/μL

11:10 PM

Ligations

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

12:05 PM

Transformation/Plating 100 μL cells, 3 μL Ligation in cuvette. electroporate and add 1 mL SOC. Recover at 37°C for 1 hour.

Plated 100 μL on 1 half of a cap plate, 50 μL on the other half of the plate at 1:10 PM

12:40 PM

PCR

Verification of insert

Per Reaction: 19 μL mastermix, 1 μL template.

Samples:

pRpoS438 colony 1

pRpoS438 colony 2

pArsRF1R2 colony 1

pArsRF1R2 colony 2

negative control (no template)

Primers:

20 micromolar

VF2

VR

Program:

3:05 PM

Gel



started new 5 mL cultures of pArsRF1R2 and LamBF1R for verification PCR tomorrow. Will re-run today's samples with new pArsR and LamBF1R samples as well as old.


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