IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/09/05

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9/5/10

10:15 am

miniprep promega kit

gas vesicles: 317.2 ng/μL

B0015: 185.5 ng/μL

B0034: 185.9 ng/μL


11:10 AM

Digestion

ArsRSDM2B0015

DNA 2 μL
H2O 39.5 μL
Buffer 4 5 μL
BSA .5 μL
XbaI 1 μL
PstI 2 μL

B0034

DNA 2.6 μL
H2O 39.9 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL

pSB1C3

DNA .64 μL
H2O 40.86 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
PstI 2 μL

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

2:45 PM

ligation

pSB1C3 digest 2 μL
ArsRSDM2B0015 digest 2 μL
B0034 digest 2 μL
H2O 11 μL
Ligase Buffer 2 μL
Ligase 1 μL

incubate at room temp for 30 min, inactivate at 80°C for 20 min

3:45 PM

Transformation

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

5:00 PM

plated 250 μL cells on Cap plate. incubate at 37°C




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