IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/09/30

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9.30.10

12:20 PM

Digestion

pSB1C3 F

DNA .62 μL
H2O 40.88 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
PstI 1 μL

pSB1C3 T

DNA 1.75 μL
H2O 39.75 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
PstI 1 μL

ArsRSDM2

DNA 1.68 μL
H2O 39.82 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
PstI 1 μL

J23100B0034ArsRSDM2B0015pSB1T3

DNA 6.54 μL
H2O 34.96 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
PstI 1 μL

Incubated at 37°C for 15 min,inactivate at 80°C for 20 min

1:15 PM

Ligation

pSB1C3F-ArsRSDM2:

pSB1C3F 2 μL
ArsRSDM2 2 μL
H2O 13 μL
Ligase Buffer 2 μL
Ligase 1 μL

pSB1C3T-ArsRSDM2:

pSB1C3T 2 μL
ArsRSDM2 2 μL
H2O 13 μL
Ligase Buffer 2 μL
Ligase 1 μL

pSB1C3F-J2334A215T:

pSB1C3F 2 μL
J2334A215T 2 μL
H2O 13 μL
Ligase Buffer 2 μL
Ligase 1 μL

pSB1C3T-J2334A215T:

pSB1C3F 2 μL
J2334A215T 2 μL
H2O 13 μL
Ligase Buffer 2 μL
Ligase 1 μL

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

2:20

Transformation

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

Plated 250 μL on Cap plates at 4:00 PM.

Also streaked out ISB169 (Cap backbone) on Cap plate.


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