IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/20

iGEM Project name 1

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dspB Track Plate result: 9 colonies Colony PCR PCR Master mix Reagent 1x rxn volume (uL) 5x rxn buffer 5 10mM dNTP 0.5 sdH2O 12.15 Phusion polymerase 0.1 MgCl2 2 DMSO - 5% 1.25 10uM fw primer (VF2) 2 10uM re primer (VR) 2 Total 25 PCR Cycles: 98C @ 3min Cycle 27x: 98C @ 10 sec 72C @ 30 sec 72C @ 40 sec 72C @ 10 min 10C @ hold Start: 12:54pm End: 2:02pm Phusion: VF2 - 64C; VR - 64C Gel verification colony PCR products Protocol: gel verification protocol in Protocol (SOP) Machine conditions: 0.5x TBE buffer, 100V, 45min Gel orientation: Gel orientation A B C D E 100bp ladder F G H I W 1 kb ladder Results: [[Image:‎|300px]] O/N culture: Protocol: SOP With 5mL of culture with chlor 1 (A,B,C,D,G,H) in 37C at 4:54pm Transformation (for His) again 9pm no colonies from yesterday changes: Use 7uL of ligation mix Use 400uL of SOC instead of LB Broth PCR using Rafael's method Attempt #4 PCR Master mix Reagent 1x rxn volume (uL) buffer 10mM dNTP 1 sdH2O 31.5 Polymerase MgCl2 2 DMSO - 5% 2.5 gDNA 1 fw rxn 2 3 re rxn 2 3 Total 45 Gave to Rafael to finish.