IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/13

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7/13/10

9:50 AM

Transformation

100ul cells, 10 ul ligation product (ArsR-pSB1A2 7/12/10). Electroporate, add 1 mL LB, recover for 1 hour at 37°C.

Plated 200 ul on Amp plates at 11:05 AM

12:00 PM

PCR

Ingredient Amount Mastermix (10 reactions)
10X buffer 2 ul 20 ul
10mM dNTPs .4 ul 4 ul
F Primer .4 ul added individually to each reaction
R Primer .4 ul added individually to each reaction
Template 1 ul (20ng) 10 ul
Polymerase .2 ul 2 ul
H2O 15.6 ul 156 ul

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

samples

LamBF1R

LamBF2R

ArsR Fusion

ArsR

pArsRF1R1

pArsRF1R2

pArsRF2R1

pArsRF2R2

Negative control (no primers)

Program

95°C 3 min
95°C 30 sec
53°C 30 sec
72°C 90 sec
go to step 2 31X
72°C 5 min
Hold at 4°C

2:35 PM

Gel

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 lamB F1R 5 μL sample, 1 μL dye
3 LamB F2R 5 μL sample, 1 μL dye
4 ArsR Fusion 5 μL sample, 1 μL dye
5 ArsR 5 μL sample, 1 μL dye
6 pArsR F1R1 5 μL sample, 1 μL dye
7 pArsR F1R2 5 μL sample, 1 μL dye
8 pArsR F2R1 5 μL sample, 1 μL dye
9 pArsR F2R2 5 μL sample, 1 μL dye
10 Negative Control 5 μL sample, 1 μL dye

Image:UIUC_7.13.10_2pm_PCR_ArsR,_ArsR-F,_pArsR.jpg‎



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