IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/18

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8/18/10

11:35 AM

Digestion

ArsRSDM2

DNA 2.7 μL (500ng)
H2O 39.8 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL

B0015

DNA 1.55 μL (500ng)
H2O 40.95 μL
Buffer 4 5 μL
BSA .5 μL
XbaI 1 μL
PstI 1 μL

pSB1T3

DNA 1 μL (500ng)
H2O 41.5 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
PstI 1 μL

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

11:50 AM

Ligation

ArsRSDM2-B0015-pSB1T3

H2O 11 μL
pSB1T3 2 μL
ArsRSDM2 2 μL
B0015 2 μL
Ligase Buffer 2 μL
Ligase 1 μL

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

12:20 PM

Streaked colonies from 8/12/10 plate since I accidentally let them sit at 37°C over the weekend and they may have lost their red color for selection. Streaked 6 colonies from each plate.

3:00 PM
5mL Culture
Made 5mL cultures (without antibiotics) of ArsR SDM2 (Amp resistant) and pSB1C3 + LamB transformation from 8/17/10

3:20 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.

4:40 PM

Plated 250 μL cells on Tet plate.