IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/03

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DspB Track Ligation See protocol: "Ligate" protocol in BioBrick Assembly Manual (in protocol binder) Changes: incubate reaction mix at RT for 20 minutes instead of 15 minutes Transformation See protocol: "Transformation" protocol (SOP) in Protocol Binder Changes: Added 10uL of ligation mix instead of 1uL; Incubate at 37C for 1 hour instead of 2 hours Tubes: A, B, C, D, E, F onto Plates: A, B, C, D, E, F, respectively Put in 37C incubator at 1630 Re-do PCR with Rafael's suggestions 2uL of MgCl2 in each PCR tube 2% and 5% DMSO for each tube Protocol: common protocols for PCR PCR Master mix Reagent 1x rxn volume (uL) Master Mix 5x rxn buffer 5 x10 50 10mM dNTP 2 x10 20 sdH2O 5.55 x10 55.5 Phusion polymerase 0.2 x10 2 MgCl2 2 x10 20 Total 14.75 147.5 PCR Tubes Label Content A dspB His 1 B dspB His 2 C dspB no His 1 D dspB no His 2 E dspB rxn1 1 F dspB rxn1 2 G dspB rxn2 1 H dspB rxn2 2 I H2O control For 1: 2% DMSO For 2: 5% DMSO To each 1 (A,C,E,G), add 2uL of fw + rev primer each and 0.5uL of DMSO and 0.75uL of H2O, and 5uL of DNA To each 2 (B,D,F,H), add 2uL of fw + rev primer each and 1.25uL of DMSO and 5uL of DNA PCR Cycles: 98C @ 2min Cycle 27x: 98C @ 10 sec 72C @ 30 sec 72C @ 40 sec 72C @ 10 min 10C @ hold Start: 1437 End: 1540 Gel Verification See protocol: gel verification protocol in Protocol binder (SOP) Changes: 1.2% agarose gel instead of 0.8% Gel orientation: Gel orientation A B C D 100bp ladder E F G H I Machine conditions: 100V, 60 min, 0.5X TBE Buffer Results: Vicki Ma 02:38, 4 August 2010 (EDT) QS Track Resuspended oligos for PCRing agrCA (with nat RBS), agrC, and agr A in the appropriate amount of sdH2O, according to idt resuspension calculator. First resuspend to 100 μM concentration (stock solution), then dilute 1/10 to 10 μM (working solution) PCR'd according to instructions that came with Phusion polymerase. PCR Master mix Reagent 1x rxn volume (uL) Master Mix 5x rxn buffer 5 x11 55 10mM dNTP 2 x11 22 sdH2O 5.55 x11 55.5 Phusion polymerase 0.05 x11 0.55 MgCl2 2 x10 12 DMSO 1 x10 11 Total 14.75 147.5 Source of DNA are colonies from a plate containing Staph aureus NCTC 8325. To each of the above 1X solutions, added 1.25 μL of each 10 μM reverse and forward primer. Did this in triplicates for each pair of primers with each PCR tube containing a different Staph aureus colony. Used the same PCR program as the dspB track (used at the same time). Gelled using 1.2% agarose over 45 minutes. No bands showed up. Water control was clear. May have to add more DNA (let colonies grow up more) and add more DMSO. Also, annealing temperature should be checked (use only part that binds to template when using Finnzyme Tm calculator). Raising DMSO concentration to 6% may also help.