IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/09/23

UIUC - Arsenic Collection System!

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9.23.10 12:05 PM PCR Verification Ingredient Amount Mastermix (6 reactions) 10X Buffer 2 μL 12 μL 10 mM dNTPs .4 μL 2.4 μL Template 1 μL (20ng) added individually to each reaction F Primer .4 μL 2.4 μL R Primer .4 μL 2.4 μL Polymerase .2 μL 1.2 μL H2O 15.6 μL 93.6 μL Primers:10 micromolar VF2, VR, ArsR F RFC10, ArsR R RFC10 (ArsR for positive control, separate reaction without verification primers, e. coli genome as template) Samples: J23100B0034ArsRSDM2B0015pSB1C3 1 J23100B0034ArsRSDM2B0015pSB1C3 2 J23100B0034ArsRSDM2B0015pSB1C3 3 + control (ArsR) - control (H2O) Program: 95°C 3 min 95°C 30 sec 55°C 30 sec 72°C 1 min go to step 2 30X 72°C 3 min hold at 4°C 12:45 PM ArsB PCR Ingredient Amount Mastermix (6 reactions) 10X Buffer 2 μL 12 μL 10 mM dNTPs .4 μL 2.4 μL Template 1 μL (20ng) 6 μL F Primer .4 μL added individually to each reaction R Primer .4 μL added individually to each reaction Polymerase .2 μL 1.2 μL H2O 15.6 μL 93.6 μL Samples: ArsB 1 ArsB 2 ArsB 3 + control (ArsR) - control (H2O) Primers: 10 micromolar ArsBF3.0 ArsBR3.0 ArsRRFC10 ArsRFRFC10 Program: 95°C 3 min 95°C 30 sec 53°C 30 sec 72°C 90 sec go to step 2 30X 72°C 3 min hold at 4°C 3:25 PM Gel Lane Sample Amount 1 1 Kb Ladder 1 μL 2 J23100B0034ArsRSDM2B0015pSB1C3 1 1 μL dye + 5 μL sample 3 J23100B0034ArsRSDM2B0015pSB1C3 2 1 μL dye + 5 μL sample 4 J23100B0034ArsRSDM2B0015pSB1C3 3 1 μL dye + 5 μL sample 5 Negative 1 μL dye + 5 μL sample 6 ArsB 1 1 μL dye + 5 μL sample 7 ArsB 2 1 μL dye + 5 μL sample 8 ArsB 3 1 μL dye + 5 μL sample 9 + control 1 μL dye + 5 μL sample 10 - control 1 μL dye + 5 μL sample 4:00 PM glycerol stocks 900 μL culture, 900 μL 50% glycerol for J23100B0034ArsRSDM2B0015pSB1C3 1 and J23100B0034ArsRSDM2B0015pSB1C3 2.