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

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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
Tubes: A, B, C, D, E, F onto Plates: A, B, C, D, E, F, respectively Put in 37C incubator at 1630
 * 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

Re-do PCR with Rafael's suggestions

 * 1) 2uL of MgCl2 in each PCR tube
 * 2) 2% and 5% DMSO for each tube


 * Protocol: common protocols for PCR


 * 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:

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.


 * 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.