IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/29

UIUC - Arsenic Collection System!

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7/29/10 10:30 AM prepared ArsR SDM1 and ArsR SDM2 samples for sequencing: 3 μL VF2 stock primer 3 μL VR stock primer 4.5 μL Template (200 ng/μL) 19.5 μL H2O 11:00 Digestions ArsR SDM1 DNA 2.4 μL H2O 40.1 μL Buffer4 5 μL BSA .5 μL EcoRI 1 μL SpeI 1 μL B0015 DNA 1.6 μL H2O 40.9 μL Buffer4 5 μL BSA .5 μL XbaI 1 μL PstI 1 μL pSB1A2 DNA 1.8 μL H2O 39.7 μL Buffer4 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. Ligations H2O 11 μL ArsR SDM 1 2 μL B0015 2 μL pSB1A2 2 μL Ligase Buffer 2 μL Ligase 1 μL incubate at room temperature for 10 min, inactivate at 80°C 20 min. 12:30 PM Transformation 100 μL cells, 3 μL Ligation, electroporate, add 1 mL SOC, recover for 1 hour at 37°C Plated 200 μL at 1:45 PM on Amp plates. 1:15 PM PCR Ingredient Amount Mastermix (4 reactions) 10X buffer 2 μL 8 μL 10mM dNTPs .4 μL 1.6 μL Template 1 μL (20 ng) 4 μL F Primer .4 μL added individually to each reaction R Primer .4 μL added individually to each reaction Polymerase .2 μL .8 μL H2O 15.6 μL 62.4 μL Per reaction: 19.2 μL mastermix, .4 μL F Primer, .4 μL R Primer. Primers: pArsR RFC10F-502 EcoRI pArsR R1 pArsR R2 pRpoS RFC0F-438 EcoRI pRpoS R Samples: pArsR F1R1 pArsR F1R2 pRpoS F-438R 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 4:00 PM Gel lane sample amount 1 1 Kb ladder 1 μL 2 pArsRF1R1 (540bp) 5 μL + 1 μL dye 3 pArsRF1R2 (547bp) 5 μL + 1 μL dye 4 pRpoS (438 bp) 5 μL + 1 μL dye 5 Tom 1 5 μL + 1 μL dye 6 Tom 2 5 μL + 1 μL dye 7 negative control 5 μL + 1 μL dye Made 5 mL culture of LamB colonies from yesterday. incubated at 37°C for miniprep tomorrow.