IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/09/19

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9.19.10

10:45 AM

Digestion

J23100B0034ArsRSDM2B0015pSB1T3

DNA 6.54 μL
H20 34.9 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
PstI 2 μL


pSB1C3

DNA .64 μL
H20 40.8 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
PstI 2 μL

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

11:00 AM

PCR

Ingredient Amount Mastermix (5 samples)
10X Buffer 2 μL 10 μL/td>
10mM dNTPs .4 μL 2 μL
Template 1 μL (20 ng) 5 μL
Primer F .4 μL Added individually to each reaction
Primer R .4 μL added individually to each reaction
Polyemerase .2 μL 1 μL
H2O 15.6 μL 78 μL

Samples:

  1. pArsR
  2. pRpoS
  3. + control ArsR
  4. - control H2O

Primers: 10 micromolar

  • pARsRF1 2.0
  • pArsRR2 2.0
  • pRpoS438 2.0
  • pRpoSR 2.0
  • ArsR F RFC10 EcoRI
  • ArsR R RFC10

Program:

11:25 AM

Ligation

95°C 3 min
95°C 30 sec
53°C 30 sec
72°C 90 sec
go to step 2 30X
72°C 5 min
hold at 4°C
J23100B0034ArsRSDM2B0015pSB1T3 2 μL
pSB1C3 2 μL
H2O 13 μL
Ligase Buffer 2 μL
Ligase 1 μL

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

12:20 PM

Transformation

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

1:35 PM

Gel

TAE, 1%, 175V

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 pARsR 1 μL dye, 5 μL sample
3 pRpoS 1 μL dye, 5 μL sample
4 Positive Control 1 μL dye, 5 μL sample
5 Negative Control 1 μL dye, 5 μL sample

Image: UIUC_9.19.10igempromotersworked!.jpg‎


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