IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/05

UIUC - Arsenic Collection System!

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7/5/10 12:15 PM PCR Ingredient Amount Mastermix (6 reactions) 10X Buffer 4 ul 24 ul 10mM dNTPs 1 ul 6 ul Template 1.56 ul (500ng) 9.36 ul Primers (F/R) 4 ul added individually to each reaction Polymerase .4 ul 2.4 ul H2O/td> 29.04 ul 174.24 ul add 36 ul mastermix and 4 ul primers to each reaction. Program: 95°C 5 min 95°C 30 sec 55°C 30 sec 72°C 2 min go to step 2 31X 72°C 5 min hold at 4°C Samples: 1. PAL 2. ArsB 3. PAL negative control (no primers) 4. ArsB negative control (no primers Ingredient Amount 10X Buffer 4 ul 10mM dNTPs 1 ul Template 1.56 ul (500 ng) F Primer 4 ul R Primer 4 ul Polymerase .4 ul H2O 25.04 ul Program: 95°C 5 min 95°C 30 sec 60°C 30 sec 72°C 2 min go to step 2 31X 72°C 5 min Hold at 4 °C Samples: 1. ArsR 2. ArsR Primers: ArsR R RFC10 ArsR F RFC10 EcoRI Gel Lane Sample Amount 1 1 Kb Ladder 1.5 ul 2 PAL 5 ul sample, 1 ul dye 3 ArsB 5 ul sample, 1 ul dye 4 PAL negative control 5 ul sample, 1 ul dye ArsB negative control 5 ul sample, 1 ul dye Ran at 175V, got a blank gel. 8:00PM Re-diluted primers to 10 micromolar, ran same PCR again. This time with two samples for ArsR as well. ran at 175 V Digestion ArsR DNA .86 ul (500 ng) H2O 41.64 ul Buffer 2 5 ul BSA .5 ul EcoRI 1 ul SpeI 1 ul pSB1A2 (6/30) DNA 1 ul H2O 41.5 ul Buffer2 5 ul BSA .5 ul EcoRI 1 ul SpeI 1 ul Incubate for 15 min at 37°C Inactivate for 20 min at 80°C 9:45 PM PCR Purification Ligation H2O 9 ul ArsR Digest 2 ul pSB1A2 Digest 2 ul 5X Ligase Buffer 4 ul Ligase 1 ul NB: used new invitrogen ligase and buffer. Incubate at room temp for 10 minutes, then inactivate at 80°C for 20 minutes. 10:30 PM Streaked out some new e. Coli on LB for colony boils 11:00 PM Transformation 100 ul cells and 5 ul ligation product, electroporated and added 1 mL LB. Recovered for 1 hour at 37°C. Plated 100 ul, 200 ul and 300 ul on separate Amp plates.