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

UIUC - Arsenic Collection System!

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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