IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/22

UIUC - Arsenic Collection System!

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7/22/10 Ordered new pRpoS438F and pArsRF1 primers so they have an EcoRI site instead o XbaI. Lawns on gas vesicle/promoter plates again No growth on pRpoS/pArsR plates 10:25 AM miniprepped ArsR/pSB1A2 colonies. concentrations: ArsR-pSB1A2 colony 1: 288.2 ng/μL ArsR-pSB1A2 colony 2: 323.9 ng/μL Diluted each sample to 20 ng/μL for PCR 11:00 AM PCR Ingredient Amount Mastermix (4 reactions) 10X Buffer 2 μL 8 μL 10 mM dNTPs .4 μL 1.6 μL Template 1 μL (20 ng) Added individually to each reaction F Primer .2 μL .8 μL R Primer .2 μL .8 μL Polymerase .2 μL .8 μL H2O 16 μL 64 μL Per reaction: 18.8 μL mastermix, .2 μL F primer, .2 μL R primer, 1 μL template Samples: ArsR-pSB1A2 colony 1 ArsR-pSB1A2 colony 2 Negative control (no template) Primers (20 micromolar) VR V2F Program: 95°C 3 min 95°C 30 sec 55°C 30 sec 72°C 90 sec go to step 2 31X 72°C 5 min hold at 4°C 2:05 PM Gel 1%, TAE 175V Lane Sample Amount 1 1 Kb Ladder 1μL 2 ArsR-pSB1A2 colony 1 5 μL sample, 1 μL dye 3 ArsR-pSB1A2 colony 2 5 μL sample, 1 μL dye 4 negative control 5 μL sample, 1 μL dye 3:15 PM Site Directed Mutagenesis PCR Modified from Quikchange kit (stratagene) Ingredient Amount 10X Buffer 5 μL 10 mM dNTPs 1 μL Template 1 μL (20ng) Primer 1 .2 μL (~125 ng) Primer 2 .2 μL (~125 ng) Polymerase 1 μL H2O 41.6 μL Sample: ArsR-pSB1A2 colony 1 Primers: ArsR SDM 1 (100 micromolar) ArsR SDM 2 (100 micromolar) Program: 95°C 3 min 95°C 30 sec 66°C 1 min 72 1 min go to step 2 12X 72 1 min hold for 4°C