IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/28

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2/28/10

Got colonies on both LamB plates!

10:00 AM

PCR

Ingredient Amount Mastermix (13 reactions)
10X Buffer 2 μL 26 μL
10mM dNTPs .4 μL 5.2 μL
Template 20 ng (1 μL) added individually to each reaction
F Primer .2 μL added individually to each reaction
R Primer .2 μL added individually to each reaction
Polymerase .2 μL 2.6 μL
H2O 16 μL 208 μL

Per Reaction: 18.6 μL mastermix, 1 μL template,.2 μL F primer, .2 μL R primer.

Primers:

20 micromolar

VF2

VR

Samples:

ArsR SDM1

ArsR SDM2

A1

A2

B1

B2

L1 RFP 1

L1 RFP 2

L3 GFP 1

L3 GFP 2

GolS

Program:

95°C 2 min
95°C 30 sec
55°C 30 sec
72°C 1 min
go to step 2 30X
72°C 2 min
hold at 4°C

10:50 AM

PCR

Ingredient Amount Mastermix (10 reactions)
10X Buffer 2 μL 20 μL
10mM dNTPs .4 μL 4 μL
Template 18 ng (.9 μL) 9 μL
F Primer .4 μL added individually to each reaction
R Primer .4 μL added individually to each reaction
Polymerase .3 μL 3 μL
DMSO 1 μL 10 μL
H2O 14.6 μL 146 μL

Per reaction: 19.2 μL mastermix, .4 μL F primer, .4 μL R primer.

Primers:

10 micromolar

ArsBF2

ArsBR2

samples:

ArsB 48°C

ArsB 51°C

ArsB 54°C

ArsB 57°C

ArsB 60°C

ArsB 63°C

ArsB 66°C

Negative control (no primers)

Program:

95°C 3 min
95°C 30 sec
48-68 gradient°C 30 sec
72°C 2 min
go to step 2 30X
72°C 5 min
hold at 4°C

1:20 PM

Gel

Top

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 ArsR SDM1 5 μL sample, 1 μL dye
3 ArsR SDM2 5 μL sample, 1 μL dye
4 A1 5 μL sample, 1 μL dye
5 A2 5 μL sample, 1 μL dye
6 B1 5 μL sample, 1 μL dye
7 B2 5 μL sample, 1 μL dye
8 L1 1 5 μL sample, 1 μL dye
9 L1 2 5 μL sample, 1 μL dye
10 L3 1 5 μL sample, 1 μL dye

Bottom:

Lane Sample Amount
1 1 Kb Ladder 5 μL sample, 1 μL dye
2 L3 2 5 μL sample, 1 μL dye
3 golS 5 μL sample, 1 μL dye
4 Negative control 5 μL sample, 1 μL dye
5 ArsB 48°C 5 μL sample, 1 μL dye
6 ArsB 51°C 5 μL sample, 1 μL dye
7 ArsB 54°C 5 μL sample, 1 μL dye
8 ArsB 57°C 5 μL sample, 1 μL dye
9 ArsB 60°C 5 μL sample, 1 μL dye
10 ArsB 63°C 5 μL sample, 1 μL dye

Image:UIUC_Sequencing.jpg‎

Lane Sample Amount
1 1 Kb Ladder 5 μL sample, 1 μL dye
2 ArsB 66°C 5 μL sample, 1 μL dye
3 Tom1 15 μL sample, 3 μL dye
4 Tom2 15 μL sample, 3 μL dye
5 Tom3 15 μL sample, 3 μL dye
6 Tom4 15 μL sample, 3 μL dye

Image:UIUC_ArsB66_and_tom's_stuff.jpg‎


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