IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/30

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7/30/10

8:20 AM

checked 5 mL culture overnights, 2 were green, 2 were normal colored.

Made glycerol stock of LamBF2R: .9 mL 50% glycerol, .9 mL culture.

8:30 AM

PCR Cleanup

promega kit

pArsRF1R2: 16 ng/μL

pRpoS438: 16 ng/μL

8:35 PM

Miniprep

Qiagen kit

LamBF2R: 68 ng/μL, 20 μL left

11:55 AM

Digestion

pRpoS438:

DNA 20 μL (320 ng)
H2O 22.5 μL
Buffer4 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL

pArsRF1R2

DNA 20 μL (320 ng)
H2O 22.5 μL
Buffer4 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL

pSB1C3

DNA .71 μL (320 ng)
H2O 41.79 μL
Buffer4 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

12:40 PM

Ligations

pRpoS438-pSB1C3

H2O 13 μL
pRpoS 2 μL
pSB1C3 2 μL
Ligase Buffer 2 μL
Ligase 1 μL

pArsRF1R2-pSB1C3

H2O 13 μL
pArsR 2 μL
pSB1C3 2 μL
Ligase Buffer 2 μL
Ligase 1 μL

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

12:45 PM

set up 5 mL cultures of ArsR-B0015-pSB1A2

1:30 PM

Transformations

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

Plated 200 μL cells and 75 μL cells on CAP plates for each sample at 2:30 PM.

1:45 PM

Miniprep

Qiagen kit

Green LamBF2R: 285 ng/μL

Forgot to make glycerol stock.