IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/12

UIUC - Arsenic Collection System!

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7/12/10 Gel Lane Sample Amount 1 LamBF1R 5 ul sample, 1 ul dye 2 LamBF2R 5 ul sample, 1 ul dye 3 ArsR Fusion 5 ul sample, 1 ul dye 4 ArsR 5 ul sample, 1 ul dye 5 pArsRF1R1 5 ul sample, 1 ul dye 6 pArsRF1R2 5 ul sample, 1 ul dye 7 pArsRF2R1 5 ul sample, 1 ul dye 8 pArsRF2R2 5 ul sample, 1 ul dye 9 negative control 5 ul sample, 1 ul dye 10 1 Kb Ladder 1 ul Faint bands not visible on picture for pArsRF2R1, pArsRF2R2, LamBF1R, LamB F2R Lost pArsRF1R, pArsRF1R2 in PCR cleanup. Eluted PCR cleanup into 2 different tubes at different volumes (50 and 20 μL): pArsRF1R1(50ul) 5.4 ng/μL pArsRF1R2 (50ul) 4.9 ng/μL ArsR (50) 12 ng/μL pArsRF1R1 (20ul) 1.8 ng/μL pArsRF1R2 (20ul) 3.2 ng/ul ArsR (20 ul) 4.8 ng/μL Digestions ArsR DNA 41.67 ul (500ng) H2O .83 ul Buffer 2 5 ul BSA .5 ul EcoRI 1 ul SpeI 1 ul pSB1A2 DNA 3.93 ul (500 ng) H2O 38.57 ul Buffer 2 5 ul BSA .5 ul EcoRI 1 ul SpeI 1 ul Incubate at 37°C for 15 minutes. Inactivate at 80°C for 20 minutes. Gel/Gel Purification: Lane Sample Amount 1 1 kB ladder 1 ul 4 pSB1A2 entire sample plus dye 5 ArsR entire sample plus dye NB: other lanes held Tom and Amanda's samples. Ran at 175V Purification yields: ArsR 56.8 ng/ul pSB1A2 8 ng/ul Ligation 1:6 ratio of vector:insert H20 13 μL pSB1A2 2 μL ArsR 2 μL 10X Ligase Buffer 2 μL Ligase 1 μL Incubate 14°C overnight, inactivate (7/13/10 9:15AM) 80°C 20 min.