IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/14

UIUC - Arsenic Collection System!

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7/14/10 11:00 AM Miniprep for plasmid backbones: pSB1A2 - RFP pSB1C3 - RFP pSB2K3 - E0033 pSB1T3 - RFP 11:10 AM PCR Ingredient Amount Mastermix (6 reactions) 10X Buffer 2 μL 12 μL 10mM dNTPs .4 μL 2.4 μL Template 1 μL (20 ng) 6 μL F Primer .4 μL added individually to each reaction R Primer .4 μL added individually to each reaction Polymerase .2 μL 1.2 μL H2O 15.6 μL 93.6 μL Per reaction: 19.2 μL mastermix, .4 μL F primer,.4 μL R primer. Samples pRpoS438 pRpoS252 ArsB Negative control (no primers) Primers pRpoSF438 pRpoSF252 pRpoSR1 ArsBF2 ArsBR2 Program 95°C 3 min 95°C 30 sec 53°C 30 sec 72°C 90 sec go to step 2 31X 72°C 5 min hold at 4°C 2:50 PM Gel Lane Sample Amount 1 1 kb Ladder 1 μL 2 pArsR 438 5 μL Sample, 1 μL dye 3 pArsR 252 5 μL Sample, 1 μL dye 4 ArsR 5 μL Sample, 1 μL dye 6:15 PM Nanodropped after PCR: pArsRF1R2: 314.6 ng/μL ArsR: 310.8 ng/μL RpoS 438: 383.4 ng/μL PCR purified samples (qiagen kit): pArsRF1R2: 24.6 ng/μL ArsR: 4.8 ng/μL RpoS 438: 12.2 ng/μL Digestions pSB1T3 DNA 1 μL (500 nog H2O 41.5 μL Buffer 2 5 μL BSA .5 μL XbaI 1 μL SpeI 1 μL pSB1T3 DNA 1 μL (500ng) H2O 41.5 μL Buffer 2 5 μL BSA .5 μL XbaI 1 μL EcoRI 1 μL pSB1C3 DNA 1.1 μL (500 ng) H2O 41.4 μL Buffer 2 5 μL BSA .5 μL XbaI 1 μL SpeI 1 μL pSB1C3 DNA 1.1 μL (500 ng) H2O 41.4 μL Buffer 2 5 μL BSA .5 μL EcoRI 1 μL SpeI 1 μL pArsRF1R1 DNA 20 μL H2O 22.5 μL Buffer 2 5 μL BSA .5 μL XbaI 1 μL SpeI 1 μL ArsR DNA 28 μL H2O 14.5 μL Buffer 2 5 μL BSA .5 μL EcoRI 1 μL SpeI 1 μL RpoS435 DNA 28 μL H2O 14.5 μL Buffer 2 5 μL BSA .5 μL XbaI 1 μL SpeI 1 μL 8:00 PM Gel/Band Extraction Zymogen Kit, 1% gel, TAE Lane Sample Amount 1 1 Kb Ladder 1 μL 2,3 pSB1T3 XbaI/SpeI each lane, 25 μL sample, 5 μL dye 4,5 pSB1T3 EcoRI/SpeI each lane, 25 μL sample, 5 μL dye 6,7 pSB1C3 XbaI/SpeI each lane, 25 μL sample, 5 μL dye 8,9 pSB1C3 each lane, 25 μL sample, 5 μL dye 10 1 Kb Ladder 1 μL Nanodrop after purification: pSB1T3 XbaI/SpeI: 28.39 ng/μL pSB1T3 EcoRI/SpeI: 20.3 ng/μL pSB1C3 XbaI/SpeI: 18.2 ng/μL pSB1C3 EcoRI/SpeI: 18.3 ng/μL SAP SAP Buffer (10X) 1 ul SAP .5 ul H2O .5 ul SAP-ed pSB1T3 XbaI/SpeI and pSB1C3 XbaI/SpeI. Incubate 20 min at 37°C, inactivate 15 min at 65°C. Flash Spin, remove DNA from top for ligations. Ligation pSB1T3 XbaI/SpeI-pArsR H2O 13 μL pSB1T3 XbaI/SpeI 2 μL pArsR 2 μL 10X Ligase Buffer 2 μL Ligase μL pSB1T3 XbaI/SpeI-RpoS H2O 13 μL pSB1T3 XbaI/SpeI 2 μL RpoS 2 μL 10X Ligase Buffer 2 μL Ligase μL pSB1T3 EcoRI/SpeI-ArsR H2O 13 μL pSB1T3 XbaI/SpeI 2 μL ArsR 2 μL 10X Ligase Buffer 2 μL Ligase μL Incubate 14°C Overnight. 80°C 20 min inactivation (7/15/10 8:45 AM)