IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/09/02

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9/2/10

12:30 PM

finished MG1655 genome prep (which Francis started 8/31).

MG1655 1: 187.6 ng/μL MG1655 2: 239.8 ng/μL

made dilutions to 20 ng/μL

1:30 PM

Digestions

ArsRSDM2 (for ligation with B0034)

DNA 1.675 μL
H2O 39.825 μL
Buffer 4 5 μL
BSA .5 μL
XbaI 1 μL
PstI 2 μL

ArsRSDM2 (for ligation with B0015)

DNA 1.675 μL
H2O 40.825 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL

B0015

DNA 1.55 μL
H2O 39.95 μL
Buffer 4 5 μL
BSA .5 μL
XbaI 1 μL
PstI 2

pSB1A2

DNA 1.82 μL
H2O 39.68 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
PstI 2 μL

pSB1K3

DNA 1.56 μL
H2O 39.94 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
PstI 1 μL

Incubated at 37°C for 15 min, inactivated at 80°C for 20 min.

2:15 PM

Ligations

ArsRSDM2-B0034-pSB1K3

H20 11 μL
ArsR SDM2 2 μL
B0034 2 μL
pSB1K3 2 μL
Ligase Buffer 2 μL
Ligase 1 μL

ArsRSDM2-B0015-pSB1K3

H20 11 μL
ArsR SDM2 2 μL
B0015 2 μL
pSB1K3 2 μL
Ligase Buffer 2 μL
Ligase 1 μL

ArsRSDM2-B0015-pSB1A2

H20 11 μL
ArsR SDM2 2 μL
B0015 2 μL
pSB1A2 2 μL
Ligase Buffer 2 μL
Ligase 1 μL

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

2:10 PM

Streaked out B0034 (ISB142) on Amp plate from glycerol stock

Streaked out B0015 (ISB147) on Amp plate and on Kan plate from glycerol stock

3:20 PM

Transformation and Plating

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

Plated 200 μL cells after 1.5 hour incubation (after 1 hour, not enough growth).

4:10 PM

'PCR

Ingredient Amount Mastermix (6 reactions)
10X Buffer 2 μL 12 μL
10 mM dNTPs .4 μL 2.4 μL
Template .9 μL (18 ng) added individually to each reaction
F Primer .4 μL 2.4 μL
R Primer .4 μL 2.4 μL
Pol .2 μL 1.2 μL
H2O 14.7 μL 88.2 μL
DMSO 1 μL 6 μL

Samples:

  • ArsB 1
  • ArsB 2
  • ArsB 3
  • ArsB 4
  • Negative control (no template)

Primers: 10 micromolar ArsBF2.0 ArsBR2.0

Program:

95°C 3 min
95°C 30 sec
53°C 30 sec
72 2 min
go to step 2 32X
72°C 5 min
hold at 4°C
°C

5:30 PM

streaked out gas vesicles (ISB157) from glycerol stock onto amp plate.