IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/19

UIUC - Arsenic Collection System!

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8/19/10 10:00 AM PCR Ingredient Amount Mastermix (4 reactions 10X Buffer 2 μL 8 μL 10 mM dNTPs .4 μL 1.6 μL Template .9 μL (18 ng) 3.6 μL F Primer .4 μL added individually to each reaction R Primer .4 μL added individually to each reaction Polymerase .3 μL 1.2 μL H2O 14.6 μL 58.4 μL DMSO 1 μL 4μL Samples: ArsB50 ArsB53 ArsB56 Negative Control (no primers) Primers: ArsBF2.0 ArsBR2.0 Program: 95°C 3 min 95°C 30 sec 50-56°C gradient 30 sec 72°C 2 min go to step 2 32X 72°C 5 min hold at 4°C 11:15 AM Made glycerol stock of LamBF2 .9 ML 50% glycerol, .9 mL LamBF2 overnight. 11:20 AM Digestion ArsRSDM2 DNA 2.7 μL (500ng) H2O 39.8 μL Buffer 4 5 μL BSA .5 μL EcoRI 1 μL SpeI 1 μL B0015 DNA 1.55 μL (500ng) H2O 39.95 μL Buffer 4 5 μL BSA .5 μL XbaI 1 μL PstI 2 μL pSB1T3 DNA 1 μL (500ng) H2O 40.5 μL Buffer 4 5 μL BSA .5 μL EcoRI 1 μL PstI 2 μL Incubate at 37 for 15°C min. Inactivate at 80°C for 20 min. 1:15 PM Ligation ArsRSDM2-B0015-pSB1T3 H2O 11 μL pSB1T3 2 μL ArsRSDM2 2 μL B0015 2 μL Ligase Buffer 2 μL Ligase 1 μL Incubate at room temperature for 10 min, then inactivate at 80°C for 20 min. 1:50 PM Transformation 50 μL cells, 2 μL ligation product. 10 min on ice, 45 sec at 42°C, 2 min on ice. then add 1 mL SOC, recover for 1 hour at 37°C shaking. 2:20 PM Gel Lane Sample Amount 1 1 Kb Ladder 1 μL 2 ArsB50 5 μL sample, 1 μL dye 3 ArsB53 5 μL sample, 1 μL dye 4 ArsB56 5 μL sample, 1 μL dye It's hard to see, but there are very faint bands around 1.3 Kb which is the expected size for ArsB Made 5 mL Cultures (2 colonies from each plate section)of the following samples: pArsRF1R2 1 pRpoS438 electroporated pRpoS438 heat shock ArsRSDM2-B0015-pSB1T3 ArsRSDM2-B0015-pSB1T3 plate had questionable looking colonies (hence re-doing yesterday's procedure today) but I picked what looked like colonies anyways. No growth on pArsRF1R2 2 or pArsRF1R2 2' plates. 3:15 PM Plating Plated 300 μL transformation product on Tet plate 3:30 PM PCR Cleanup ArsB50: 4.8 ng/μL ArsB53: 6.1 ng/μL ArsB56: 6.3 ng/μL Francis Miniprepped ArsRSDM2 and LamBF2 overnights, both at 250 ng/μL.