IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/20

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7/20/10

10:50 AM

checked plates from yesterday, got lawns. inoculated 5 mL borth for each plate, incubating in 37°C room until tonight. Tomorrow will miniprep/PCR to see if insert actually in plasmid. Also made new Kan plates and will replate on these since there really shouldn't have been lawns this morning.

11:30 AM

PCR clean up of yesterday's samples:

pRpoS 438 30.5 ng/μL

ArsR 25 ng/μL

pArsR 25.5 ng/μL

2:35 PM

Digestions

ArsR

DNA 17 μL
H2O 25.5 μL
Buffer2 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL

pSB1A2

DNA 1.82 μL
H2O 40.68 μL
Buffer2 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.

3:15 PM

Ligations

ArsR-pSB1A2

H2O 13 μL
ArsR 2 μL
pSB1A2 2 μL
Ligase Buffer 2 μL
Ligase 1 μL

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

4:15 PM

Transformations

100 μL cells, 3 μL ligation product. electroporate, add 1 mL LB, recover at 37°C for 1 hour.

100 μL NEB cells, 3 μL ligation product. electroporate, add 1 mL LB, recover at 37°C for 1 hour.

5:15 PM

Plating

200 ul on Amp plate



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