IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/09/19

UIUC - Arsenic Collection System!

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9.19.10 10:45 AM Digestion J23100B0034ArsRSDM2B0015pSB1T3 DNA 6.54 μL H20 34.9 μL Buffer 4 5 μL BSA .5 μL EcoRI 1 μL PstI 2 μL pSB1C3 DNA .64 μL H20 40.8 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. 11:00 AM PCR Ingredient Amount Mastermix (5 samples) 10X Buffer 2 μL 10 μL/td> 10mM dNTPs .4 μL 2 μL Template 1 μL (20 ng) 5 μL Primer F .4 μL Added individually to each reaction Primer R .4 μL added individually to each reaction Polyemerase .2 μL 1 μL H2O 15.6 μL 78 μL Samples: pArsR pRpoS + control ArsR - control H2O Primers: 10 micromolar pARsRF1 2.0 pArsRR2 2.0 pRpoS438 2.0 pRpoSR 2.0 ArsR F RFC10 EcoRI ArsR R RFC10 Program: 11:25 AM Ligation 95°C 3 min 95°C 30 sec 53°C 30 sec 72°C 90 sec go to step 2 30X 72°C 5 min hold at 4°C J23100B0034ArsRSDM2B0015pSB1T3 2 μL pSB1C3 2 μL H2O 13 μL Ligase Buffer 2 μL Ligase 1 μL Incubate at room temperature for 15 min, inactivate at 80°C for 20 min 12:20 PM Transformation 50 μL cells, 2 μL ligation, 10 min on ice, 45 sec at 42°C, 2 min on ice. add 1 mL LB, recover shaking at 37°C for 1 hour. 1:35 PM Gel TAE, 1%, 175V Lane Sample Amount 1 1 Kb Ladder 1 μL 2 pARsR 1 μL dye, 5 μL sample 3 pRpoS 1 μL dye, 5 μL sample 4 Positive Control 1 μL dye, 5 μL sample 5 Negative Control 1 μL dye, 5 μL sample