IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/05

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dspB Track

 * Transformants from last day (Aug 04)

Colony PCR
Protocol: See common protocol Changes: use Phusion pol
 * Colony PCR plates 2 + water control (W)
 * To 3 and water, add 2uL of fw + rev primer each, total 4uL of primers
 * To 3 and water, add G1004 (77.12C) & G1005 (75.38C) [Tm calculated using Finnzymes.com calculator)
 * Pick colony from respective plates to respective tubes

PCR Cycles:
 * 98C @ 3min
 * Cycle 27x:
 * 98C @ 10 sec
 * 72C @ 30 sec
 * 72C @ 40 sec
 * 72C @ 10 min
 * 10C @ hold

Start: 1318 End: 1421

Gel verification for colony PCR products
Gel orientation: Results:
 * Protocol: gel verification protocol in Protocol (SOP)
 * Changes: 1.2% agarose gel
 * Machine conditions: 0.5x TBE buffer, 100V, 45min

Restriction Digest

 * On new PCR samples, try both iGEM and biobrick RD + Ligations
 * Vicki: iGEM; Marianne: biobrick

Vicki: Protocol from SOP
 * Changes: incubation time 1 hour instead of 2 hours


 * DNA: add 5uL of each C and D and psb1A3 (c,d)
 * Enzymes: EcoRI and Pst I (1uL of each to each tube)

Marianne: Protocol from biobrick (RD)
 * Supermix - same as SOP supermix
 * DNA: add 5uL of each A,B, and psb1A3 (a,b)
 * Enzymes: EcoRI and PstI (1uL each)
 * Changes: 30 min incubation instead of 15 min

Gel verification on RD
Gel orientation:
 * Protocol: biobrick "digest" protocol
 * Changes: run 10uL instead of 20uL

Ligations
Calculations $$ ratio \times \frac{insert \rm length}{vector \rm length} \times vector \rm mass = insert \rm mass (ng)$$ $$3 \times \frac{1200}{2400} \times =  ng$$ $$1uL \rm vector\times\frac{ng \rm vector}{1uL vector}\times\frac{ng \rm insert}{ng \rm vector}\times\frac{uL \rm insert}{x ng \rm insert} = $$
 * Where x = concentration of insert

Transformation
Vicki Ma 20:24, 10 August 2010 (EDT)
 * Protocol: Transformation protocol from SOP
 * Changes: 10uL of ligation mix instead of 1uL; 1 hour incubation instead of 2 hours
 * Transformants in 37C @ 1815 (3&4)
 * Transformants in 37C @ 1715 (1&2)