IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/19

UIUC - Arsenic Collection System!

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7/19/10 10:00 AM Francis finished up genomic prep for me using the DNeasy kit. got a concentration of 98 ng/μL diluted to 100 μL aliquots at 20ng/μL 3:20 PM PCR Ingredient Amount Mastermix (14 reactions) 10X buffer 2 μL 28 μL 10 mM dNTPs .4 μL 5.6 μL Template 20 ng (1 μL) 14 μL F Primer .4 μL 5.6 μL R Primer .4 μL 5.6 μL Polymerase .2 μL 2.8 μL H2O 15.6 μL 218.4 μL Per Reaction: 19.2 μL mastermix, .4 μL F primer, .4 μL reverse primer samples pRpoS438 pRpoS232 ArsB LamB F1R LamB F2R ArsR fusion ArsR pArsR F1R1 pArsR F1R2 pArsR F2R1 pArsR F2R2 Negative Control (no primers) Primers pRpoSF438 pRpoSF232 pRpoSR ArsBF2 ArsBR2 ArsR F RFC10 EcoRI ArsR R RFC10 ArsR R fusion BSAI ArsR F fusion BSAI pARsR F1 pArsR F2 pArsR R1 pArsR R2 LamB F1 LamB F2 LamB R 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 6:30 PM Gel Top: Lane Sample Amount 1 forgot to load 1 kB ladder N/A 2 pRpoS438 5 μL sample, 1 μL dye 3 pRpoS 5 μL sample, 1 μL dye 4 ArsB 5 μL sample, 1 μL dye 5 LamB F1R 5 μL sample, 1 μL dye 6 LamB F2R 5 μL sample, 1 μL dye 7 ArsR Fusion 5 μL sample, 1 μL dye 8 ArsR 5 μL sample, 1 μL dye 9 pArsRF1R1 5 μL sample, 1 μL dye 10 pArsRF2R2 5 μL sample, 1 μL dye Lane Sample Amount 1 forgot to load 1 Kb ladder N/A 2 pArsR F2R1 5 μL sample, 1 μL dye 3 pArsR F2R2 5 μL sample, 1 μL dye 4 negative control 5 μL sample, 1 μL dye 5 OmpA (Francis) 5 μL sample, 1 μL dye 6 Francis' Sample 5 μL sample, 1 μL dye 7 Francis' Sample 5 μL sample, 1 μL dye 7:00 PM Digestions J23100 DNA .5 μL H2O 42 μL Buffer 2 5 μL BSA .5 μL EcoRI 1 μL SpeI 1 μL J23119 DNA 2.64 μL H2O 39.86 μL Buffer 2 5 μL BSA .5 μL EcoRI 1 μL SpeI 1 μL gas vesicles DNA 1.16 μL H2O 41.34 μL Buffer 2 5 μL BSA .5 μL XbaI 1 μL PstI 1 μL pSB1K3 DNA 1.56 μL H2O 40.94 μL Buffer 2 5 μL BSA .5 μL EcoRI 1 μL PstI 1 μL incubate at 37°C for 15 min, inactivate at 80°C for 20 min. 8:20 Ligations J23100-gas vesicles-pSB1K3 H2O 11 μL J23100 2 μL Gas vesicles 2 μL pSB1K3 2 μL Ligase Buffer 2 μL Ligase 1 μL J23119-gas vesicles-pSB1K3 H2O 11 μL J23119 2 μL Gas vesicles 2 μL pSB1K3 2 μL Ligase Buffer 2 μL Ligase 1 μL incubate at room temp for 10 min, inactivate at 80°C for 20 min. 9:15 PM Transformations and Plating 100 μL cells, 3 μL ligation product, electroporate, add 1 mL LB, recover at 37°C for 1 hour. Plated 200 μL cells on Kan plates at 10:15 PM.