IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/03

UIUC - Arsenic Collection System!

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8/3/10 9:45 AM Digestions ArsR-B0015 DNA 2 μL H2O 39.5 μL Buffer 4 5 μL BSA .5 μL XbaI 1 μL PstI 2 μL B0034 DNA .54 μL H2O 41.96 μL Buffer 4 5 μL BSA .5 μL EcoRI 1 μL SpeI 1 μL pSB1C3 DNA 1.11 μL H2O 40.4 μL Buffer 4 5 μL BSA .5 μL EcoRI 1 μL PstI 2 μL Incubate at 37°C for 15 min, inactivate at 80°C for 20 min. 10:45 AM Miniprep Promega Kit Concentrations: pRpoS438 colony 1: 214.4 ng/μL pRpoS438 colony 2: 380.3 ng/μL pArsRF1R2 colony 1: 246.9 ng/μL pArsRF1R2 colony 2: 456.2 ng/μL 11:10 PM Ligations ArsR-B0015 2 μL B0034 2 μL pSB1C3 2 μL H2O 11 μL Ligase Buffer 2 μL Ligase 1 μL Incubate at room temperature for 10 min, inactivate at 80°C for 20 min. 12:05 PM Transformation/Plating 100 μL cells, 3 μL Ligation in cuvette. electroporate and add 1 mL SOC. Recover at 37°C for 1 hour. Plated 100 μL on 1 half of a cap plate, 50 μL on the other half of the plate at 1:10 PM 12:40 PM PCR Verification of insert Ingredient Amount Mastermix (6 reactions) 10X Buffer 2 μL 12 μL 10mM dNTPs .4 μL 2.4 μL Template 1 μL (20 ng) added individually to each reaction F Primer .2 μL 1.2 μL R Primer .2 μL 1.2 μL Polymerase .2 μL 1.2 μL H2O 16 μL 96 μL Per Reaction: 19 μL mastermix, 1 μL template. Samples: pRpoS438 colony 1 pRpoS438 colony 2 pArsRF1R2 colony 1 pArsRF1R2 colony 2 negative control (no template) Primers: 20 micromolar VF2 VR 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 3:05 PM Gel Lane Sample Amount 1 1 Kb Ladder 1 μL 2 pRpoS438 1 5 μL sample, 1 μL dye 3 pRpoS438 2 5 μL sample, 1 μL dye 4 pArsRF1R2 1 5 μL sample, 1 μL dye 5 pArsRF1R2 2 5 μL sample, 1 μL dye 6 negative control 5 μL sample, 1 μL dye 7-10 Tom/Amanda's samples N/A started new 5 mL cultures of pArsRF1R2 and LamBF1R for verification PCR tomorrow. Will re-run today's samples with new pArsR and LamBF1R samples as well as old.