IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/25

iGEM Project name 1

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dspB Track O/N culture taken out at 1003 Transformants taken out at 1003 Results: 1,4,5,8,11,14,15,his,no-his: TMTC Results: AA,TB: no colonies Colony PCR PCR Master mix Reagent 1x rxn volume (uL) Master Mix 5x rxn buffer 5 x15 75 10mM dNTP 0.5 x15 7.5 sdH2O 12.15 x15 182.25 Phusion polymerase 0.1 x15 1.5 MgCl2 2 x15 30 DMSO - 5% 1.25 x15 18.75 10uM fw primer (G1004) 2 x15 30 10uM re primer (G1005) 2 x15 30 Total 25 375 PCR Tubes: 1,4,5,8,11,14,15,his,n1,n2,n3,n4,n5,W(H2O control) 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: 1148 End: 1251 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 1 4 5 8 11 14 15 100 bp ladder his n1 n2 n3 n4 n5 W n2 Results: 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 100 bp ladder (quick load) 1 4 5 8 11 14 15 100 bp ladder(iGEM) his n1 n2 n3 n4 n5 W n2 100 bp ladder (Quick load) Results: Restriction Digest Chlor backbone psb1C3 Protocol: biobrick (digesting) Restriction Digest Supermix REAGENTS 1 RXN VOLUME (uL Buffer 2 5 BSA 0.5 ddH2O 37 Total 42.5 Add: 5uL of backbone + 5uL of insert Enzymes: EcoRI and PstI (1uL each to each tube) Biofilm Track Eric F. + Melody Biofilm Growth Protocol Day 3 (100823E + M) 100823E removed from incubator @ 1100 Controls showed no growth (woot woot) Followed procedure and placed in biosafet cabinet overnight 100823M removed from incubator @ 1335 Growth in control wells C4, C6, D10 Some showed a distinct colony Contamination in growth wells C9, E5, D9 Black dots - looked like growth of some other bacteria Biofilm Growth Protocol Day 2 (100824E + M) Removed O/N cultures from incubator @ 1100 (20 hrs growth) Plate layout below: 100824E + M Plate Layout Row 3 4 5 6 7 8 9 10 C C 8 C 8 R C R C D 8 C 8 8 R C R C E 8 8 C C C R C R Note: Controls chosen at random R = RN4220 8 = 8325-4 C = control 100824M into incubator @ 1635 100824E into incubator @ 1642 100824E-E10 not fully filled, disregard all data from it