IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/13

iGEM Project name 1

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dspB Track Plate results: 50uL HR - no colonies 450uL HR - ~20 colonies (most of them red); (A, D - white?; B, C - pink?) 50uL HC - no colonies 450uL NC - no colonies 50uL NR - 3 colonies (2red) (A) 450uL NR - ~20 colonies (~ half of them red) (A, B, C, D) 50uL NC - colonies 450uL NC - 34 colonies (A, B, C, D) Colony PCR PCR Master mix Reagent 1x rxn volume (uL) Master Mix 5x rxn buffer 5 x16 80 10mM dNTP 0.5 x16 8 sdH2O 12.15 x16 194.4 Phusion polymerase 0.1 x16 1.6 MgCl2 2 x16 32 DMSO - 5% 1.25 x16 20 10uM fw primer (G1004) 2 x16 32 10uM re primer (G1005) 2 x16 32 Total 25 400 Pick colony from respective plate and place in respective tubes PCR Cycles: 98C @ 3min Cycle 27x: 98C @ 10 sec 72C @ 30 sec 72C @ 40 sec 72C @ 10 min 10C @ hold Start: 1:53pm End: 2:57pm Gel verification colony PCR products Protocol: gel verification protocol in Protocol (SOP) Changes: 0.6g agarose/70mL 0.5x TBE buffer Machine conditions: 0.5x TBE buffer, 100V, 50min Gel orientation: Gel orientation 450uL NR A 450uL NR B 450uL NR C 450uL NR D 50uL NR A 450uL NC A 450uL NC B 100 bp ladder 450uL NC C 450uL NC D 450uL HR A 450uL HR B 450uL HR C 450uL HR D W Results: Re-Gel verification colony PCR products Protocol: gel verification protocol in Protocol (SOP) Changes: 0.6g agarose/70mL 0.5x TBE buffer Machine conditions: 0.5x TBE buffer, 100V, 50min Gel orientation: Gel orientation 450uL NC A 450uL NC B 450uL NC C 450uL NC D 50uL HR A 450uL HR B 450uL HR C 100 bp ladder 450uL HR D C 450uL NR A 450uL NR B 450uL NR C 450uL NR D 50uL NR A W 1kb ladder Results: Began O/N Culture in 37C shaking incubator at 11:30pm