IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/09/02

UIUC - Arsenic Collection System!

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9/2/10 12:30 PM finished MG1655 genome prep (which Francis started 8/31). MG1655 1: 187.6 ng/μL MG1655 2: 239.8 ng/μL made dilutions to 20 ng/μL 1:30 PM Digestions ArsRSDM2 (for ligation with B0034) DNA 1.675 μL H2O 39.825 μL Buffer 4 5 μL BSA .5 μL XbaI 1 μL PstI 2 μL ArsRSDM2 (for ligation with B0015) DNA 1.675 μL H2O 40.825 μL Buffer 4 5 μL BSA .5 μL EcoRI 1 μL SpeI 1 μL B0015 DNA 1.55 μL H2O 39.95 μL Buffer 4 5 μL BSA .5 μL XbaI 1 μL PstI 2 pSB1A2 DNA 1.82 μL H2O 39.68 μL Buffer 4 5 μL BSA .5 μL EcoRI 1 μL PstI 2 μL pSB1K3 DNA 1.56 μL H2O 39.94 μL Buffer 4 5 μL BSA .5 μL EcoRI 1 μL PstI 1 μL Incubated at 37°C for 15 min, inactivated at 80°C for 20 min. 2:15 PM Ligations ArsRSDM2-B0034-pSB1K3 H20 11 μL ArsR SDM2 2 μL B0034 2 μL pSB1K3 2 μL Ligase Buffer 2 μL Ligase 1 μL ArsRSDM2-B0015-pSB1K3 H20 11 μL ArsR SDM2 2 μL B0015 2 μL pSB1K3 2 μL Ligase Buffer 2 μL Ligase 1 μL ArsRSDM2-B0015-pSB1A2 H20 11 μL ArsR SDM2 2 μL B0015 2 μL pSB1A2 2 μL Ligase Buffer 2 μL Ligase 1 μL Incubate at room temp for 15 min, inactivate at 80°C for 20 min 2:10 PM Streaked out B0034 (ISB142) on Amp plate from glycerol stock Streaked out B0015 (ISB147) on Amp plate and on Kan plate from glycerol stock 3:20 PM Transformation and Plating 100 μL cells, 3 μL ligation product, electroporate. recover in 1 mL of LB at 37°C for 1 hour Plated 200 μL cells after 1.5 hour incubation (after 1 hour, not enough growth). 4:10 PM 'PCR Ingredient Amount Mastermix (6 reactions) 10X Buffer 2 μL 12 μL 10 mM dNTPs .4 μL 2.4 μL Template .9 μL (18 ng) added individually to each reaction F Primer .4 μL 2.4 μL R Primer .4 μL 2.4 μL Pol .2 μL 1.2 μL H2O 14.7 μL 88.2 μL DMSO 1 μL 6 μL Samples: ArsB 1 ArsB 2 ArsB 3 ArsB 4 Negative control (no template) Primers: 10 micromolar ArsBF2.0 ArsBR2.0 Program: 95°C 3 min 95°C 30 sec 53°C 30 sec 72 2 min go to step 2 32X 72°C 5 min hold at 4°C °C 5:30 PM streaked out gas vesicles (ISB157) from glycerol stock onto amp plate.