IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/08

UIUC - Arsenic Collection System!

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7/8/10 11:00 AM Colony Boil 25 ul H2O, swiped across plate to pick up a bunch of colonies. incubate at 95°C for 2 min. nanodropped, 444.6 ng/ul. PCR Ingredient Amount per reaction Mastermix (9 reactions) 10X buffer 4 ul 36 ul 10 mM dNTPs 1 ul 9 ul Template 1.12 ul (500ng) 10.08 ul F Primer 4 ul added individually to each reaction R Primer 4 ul added individually to each reaction Polymerase .4 ul 3.6 ul H2O 25.48 ul 229.32 ul Per Reaction: 32 ul Mastermix, 4 ul F Primer, 4 ul R Primer Samples: pArsR F1R1(540 bp) pArsR F1R2 (547 bp) pArsR F2R1 (293 bp) pArsR F2R2 (300 bp) ArsR 1 ArsR 2 negative control (no primers) Primers (10mM): pArsR F1 pArsR F2 pArsR R1 pArsR R2 ArsR F RFC10 EcoRI ArsR R RFC10 Program: 95°C 2 min 95°C 30 sec 55°C 30 sec 72°C 1 min go to step 2 31X 72°C 5 min hold at 4°C 2:05 PM gel Lane Sample Amount 1. 1 Kb Ladder 1 ul 2. pArsR F1R1 5ul sample, 1 ul dye 3. pArsR F1R2 5ul sample, 1 ul dye 4. pArsR F2R1 5ul sample, 1 ul dye 5. pArsR F2R2 5ul sample, 1 ul dye 6. ArsR1 5ul sample, 1 ul dye 7. ArsR2 5ul sample, 1 ul dye 8. negative control 5ul sample, 1 ul dye 90V Blank gel again 2:30 PM PCR #a million of LamB and ArsR and pArsR - NEW MIXTURE Ingredient Amount per 20ul reaction Mastermix (9 reactions) 10X buffer 2 ul 18 ul 10 mM dNTPs .4 ul 3.6 ul Template 1 ul (20ng) 9 ul 10uM F Primer .4 ul added individually to each reaction 10uM R Primer .4 ul added individually to each reaction Pfu Turbo DNA Polymerase .2 ul 31.8 ul H2O 15.6 ul 140.4 ul Per Reaction: 32 ul Mastermix, 4 ul F Primer, 4 ul R Primer Samples: 1. LamB F1R 2. LamB F2R 3. ArsR 4. pArsR F1R1(540 bp) 5. pArsR F1R2 (547 bp) 6. pArsR F2R1 (293 bp) 7. pArsR F2R2 (300 bp) 8. negative control (no primers) Program: 95°C 3 min 95°C 30 sec 55°C 30 sec 72°C 90 sec go to step 2 31X 72°C 5 min hold at 4°C The gel for this PCR was run on Monday July 12th 3:25 digestions pArsR DNA 10 ul (500 ng) H2O 32.5 ul Buffer 2 5 ul BSA .5 ul SpeI 1 ul XbaI 1 ul pSB1T3 DNA 1 ul (500 ng) H2O 41.5 ul Buffer 2 5 ul BSA .5 ul SpeI 1 ul XbaI 1 ul Incubate at 37°C for 15 min, inactivate at 80°C for 20 min PCR purified pSB1T3 and SAP: 2 ul SAP buffer, 1 ul SAP 37°C 10 min, 65°C 15 min. 4:45 PCR Ingredient Amount per reaction Mastermix (9 reactions) 10X Buffer 4 ul 36 ul 10 mM dNTPs 1 ul 9 ul Template 1.12 ul (500 ng) 10.08 ul F Primer 4 ul added individually to each reaction R Primer 4 ul added individually to each reaction Pol .4 ul 3.6 ul H2O 25.48 ul 229.32 ul Per Reaction: 32 ul mastermix, 4 ul F primer, 4 ul R primer Primers: pArsRF1 pArsRF2 pArsRR1 pArsRR2 ArsR F RFC10 EcoRI ArsR R RFC10 Samples: pArsR F1R1 540bp pArsR F1R2 547bp pArsR F2R1 293 bp pArsR F2R2 300 bp ArsR Negative control (no primers Program 95°C 5 min 95°C 30 sec 55°C 30 sec 72°C 1 min go to step 2 31X 72°C 5 min hold at 4°C SAP Backbone 2.6 ul SAP buffer 1.3 ul SAP Incubate at 37°C for 10 min, inactivate at 65°C for 15 min. Gel Lane Sample Amount 1 1 Kb Ladder 1 ul 2 Empty N/A 3 Empty N/A 4 pArsR (7/8/10) 50 ul sample, 10 ul dye 5 Empty N/A 6 pSB1T3/SAP (7/8/10) 30 ul sample, 6 ul dye Lane Sample Amount 1 pSB1T3/SAP (7/1/10) 50 ul sample, 10 ul dye 2 Empty N/A 3 pArsR (7/7/10) 50 ul sample, 10 ul dye 4 Empty N/A 5 Empty N/A 6 1 Kb Ladder 1 ul 8:40 PM Band Extraction Zymogen Kit pSB1T3 (7/1) and pArsR (7/7). the other samples didn't show up on gel. Concentrations post-band extraction: pSB1T3 8 ng/ul pArsR 20 ng/ul 9:20 PM Ligations H2O 13 ul pSB1T3 2 ul pArsR 2 ul 10X Ligase Buffer 2 ul Ligase 1 ul Incubate at 14°C overnight, inactivate at 80°C for 20 min tomorrow.