IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/26
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8/26/109:00 AM Dilutions to 20 ng/ul
9:35 AM Verification PCR
Per Reaction: 19 μL mastermix, 1 μL template Samples:
Primers: VF2, VR at 10 micromolar Program:
12:50 PM Digestion B0015:
ArsRSDM2
pSB1T3
Incubate at 37°C for 15 min, inactivate at 80°C for 20 min. 1:00 PM PCR ArsB
Samples:
Primers: ArsB F 2.0, ArsB R 2.0 at 10 micromolar Program:
2:00 PM Gel
Ligation
incubate at room temperature for 10 min, inactivate at 80°C for 20 min. 2:35 PM Transformation 50 μL cells, 2 μL ligation product. 10 min on ice, 45 sec at 42°C, 2 min on ice. add 1 mL LB, recover for 1 hour at 37°C shaking. 3:10 PM PCR Cleanup Qiagen kit pRpoS438SDM2 2: 21.6 ng/μL pArsRF1R2SDM2 1: 21ng/μL pArsRF1R2SDM2 2: 22.7 ng/μL 3:45 PM Sequencing reactions
4:00 PM plated 200 μL transformation on tet plate
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