IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/26

UIUC - Arsenic Collection System!

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7/26/10 9:50 AM Made glycerol stocks of 5 mL cultures ArsR SDM1 ISB206 ArsR SDM2 ISB207 Miniprep (promega kit) ArsR SDM1 184.9 ng/μL ArsR SDM2 209.3 ng/μL 12:15 PM PCR Ingredient Amount Mastermix (4 reactions) 10X Buffer 2 μL 8 μL 10mM dNTPs .4 μL 1.6 μL Template .9 μL (18ng) 3.6 μL F Primer .4 μL added individually to each reaction R Primer .4 μL added individually to each reaction Polymerase .3 μL 1.2 μL DMSO 1 μL 4 μL H2O 14.6 μL 58.4 μL Per Reaction: 19.2 μL mastermix, .4 μL F primer, .4 μL R primer. Samples LamBF1R LamBF2R ArsB Negative control (no primers) Primers All primers at 10 micromolar LamBF1 LamBF2 LamBR ArsBF2 ArsBR2 Program 95°C 3 min 95°C 30 sec 53°C 30 sec 72°C 2 min go to step 2 31X 72°C 5 min hold at 4°C 3:30 PM Gel 1% TAE, 175V Lane Sample Amount 1 1 Kb Ladder 1 μL 2 ArsB 5μL sample + 1μL dye 3 LamBF1R 5μL sample + 1μL dye 3 LamBF2R 5μL sample + 1μL dye 4 Negative Control 5μL sample + 1μL dye