IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/06

iGEM Project name 1

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7/6/10 11:00 AM At this point, we ran a gradient PCR of Pal and one ArsR sample. It didn't work at all - no bands on the gel, except the ladder. The extension time was 1 minute with 30 cycles. 95 - 2min (95 - 30sec; 55ish - 30sec; 72 - 1min | 30x) 72 - 1 min 4 - hold Rxn Mixture Component ul 7x MM 10x Buffer 4 28 10mM dNTP 1t 37 Template (686ng/ul colony boil of E. Coli .8ul 5.6 10uM F Primer 4 -- 10uM R Primer 4 -- Pfu Turbo DNA Pol .4 2.8 H2O 25.8 224 Samples: 1 PAL Annealing Temp - 47C 2 PAL Annealing Temp - 50C 3 PAL Annealing Temp - 52C 4 PAL Annealing Temp - 55C 5 ArsR Annealing Temp - 55C 6 Neg Control - H20 instead of Primers - 55C Run at 100V for 30min - No Bands (except the ladder) 12:00 PM Gel of Digests Lane Sample Amount 2 ArsR digest 35 ul sample, 7 ul dye 8-10 pSB1A2 Digest 35 ul sample, 7 ul dye 6 1 Kb Ladder 1 ul Run at 90V PCR Ingredient Amount Mastermix (9 reactions) 10X buffer 4 ul 36 ul 10mM dNTPs 1 ul 9 ul Template .8 ul (500 ng) 7.2 ul F Primer 4 ul individually added to each reaction R Primer 4 ul individually added to each reaction Polymerase .4 ul 3.6 ul H2O 25.8 ul 232.2 ul Each reaction gets 32 ul mastermix, 4 ul of each primer. Primers: PAL F/R ArsR R RFC10 ArsR F RFC10 EcoRI Samples: 1. PAL47°C 2. PAL50°C 3. PAL52°C 4. PAL55°C 5. PAL60°C 6. PAL60°C 7. negative control, no primers Program: 95°C 2 min 95°C 30 sec Gradient 30 sec 72°C 1 min go to step 2 31X 72°C 5 min hold at 4°C 2:40 PM Band extraction of pSB1A2 from gel 3:15 PM re-streaked e. Coli from yesterday for 2nd overnight for colony boils. 7:00 PM Put transformations from 7/6 12am - Erin into 4C room - No Colonies Everyday is a Monday in the life of Erin Insert your content here. No I don't want to. Go to hell. Hi. My name is Erin. I am also referred to as, "The Bork", "Borky" and "PB & J". Although I never warmed to these terms of affection, I understand that they do reflect my persona and thus my essence, very well. I am very tired. It is during this time, when I'm sleep deprived, overworked, sore, and stressed, when I begin to lose myself in various philosophical quandaries. Today's topic of interest does not lie in the archaic ramblings of Plato or Aristotle, but rather in the quarky, uncanny ability of Steve's hairline to recede to his occiput. How such a phenomenon occurs has reduced my intellectual capacitance to mere shapes: squares, circles, you get the point. Every day, it's like watching the waves rise of the shore, leaving a residual bump, scarcely noticeable, save for the keen of eye. But he's begun to grow his hair out, which has worked only to thwart my amusement. I'll wait till he gets a haircut. Wow. Erin / Francis ... nice. (from Meagan)