IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/31

iGEM Project name 1

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Biofilm Track Melody Took out O/N cultures of RN4220 and 8325-4 from non-shaking incubator @ 11:52 a.m. Biofilm Protocol Day 2 Innoculated plates 100830M120+100830M222 100830M1 + 100830M2 96-well plate Layout Row 3 4 5 6 7 8 9 10 C R R C R 8 8 8 C D R R R R C 8 8 8 E C R R C 8 8 8 C Note: Controls chosen at random R = RN4220 8 = 8325-4 C = control Incubated @ 2:00 p.m. w/o shaking Overnight cultures 2 O/N cultures of RN4220 and 8325-4 one in incubator w/ shaking one in incubator w/o shaking Both in @ 3:57 p.m. DspB Track PCR off gDNA for more PCR products PCR Master mix Reagent 1x rxn volume (uL) Master Mix 5x rxn buffer 5 x10 50 10mM dNTP 0.5 x10 5 sdH2O 7.15 x10 71.5 Phusion polymerase 0.1 x10 1 MgCl2 2 x10 20 DMSO - 5% 1.25 x10 12.5 Total 16 160 PCR Tubes Tube Content His1,His2,His3,His4 dspB his no his1,no his2,no his3,no his4 dspB no-his W (H2O control) To His1,His2,His3,His4: add 2uL of up-his & dw primers each To no his1,no his2,no his3,no his4: add 2uL of up & dw primers each To all, add 5uL of DNA PCR Cycles: 98C @ 30s Cycle 27x: 98C @ 10 sec 72C @ 30 sec 72C @ 40 sec 72C @ 10 min 10C @ hold Start: 1234 End: 1335 Gel verification colony PCR products Protocol: gel verification protocol in Protocol (SOP) Changes: 1% agarose gel Machine conditions: 0.5x TBE buffer, 100V, 60min Gel orientation: Gel orientation his1 his2 his3 his4 100bp ladder no his1 no his2 no his3 no his4 W (control) Results: PCR purification All 4 tubes of His in 1 spin column (88uL + 440uL PBI) All 4 tubes of no his in 1 spin column (88uL + 440uL PBI) [His] = 126.3ng/uL [No his] = 196.8ng/uL