IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/01

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Melody

 * Took O/N cultures out of shaking and non-shaking incubator @ 10:00 a.m.
 * No contamination in both controls


 * Took out plate 100830M120 @ 10:00 a.m.
 * No contamination


 * Took out plate 100830M222 @ 12:00 p.m.
 * contamination in D7 (characteristic white colonies)

Biofilm Protocol Day 3

 * 3 x 300 ul PBS wash on plates 100830M120 + 100830M222
 * 100830M120 heat fixed @ 11:55 a.m.
 * 100830M222 heat fixed @ 12:50 p.m.

Biofilm Protocol Day 2

 * Innoculate into plates 100831M24 + 100831M26

100831M24 + 100831M26 Plate Layout

 * randomized controls


 * 100831M26 incubated @ 2:30 p.m.
 * 100831M24 incubated @ 3:20 p.m.

Biofilm Day 4 continued

 * Initial OD550 readings on 100824E + 100824M

100824E Data

 * Note: Red = Control

100824M Data

 * Note: Red = Control


 * Resolubilized in 95% ethanol

100824ERS

 * Note: Red = Control

100824MRS

 * Note: Red = Control

Biofilm Protocol Day 1

 * O/N cultures of RN4220 and 8325-4 incubated @ 4:40 p.m. w/o shaking

TSA plates

 * TSA plate spread with RN4220 and 8325-4
 * Incubated @ 5:03 p.m.

Autoclaved Tips

 * 300ul, 1000ul, 10ul pipette tips autoclaved

Restriction Digest

 * DNA, add...
 * His: 9.5uL
 * No his: 9.5uL
 * RFP chlor bb: 7uL
 * ccdB chlor bb: 7uL


 * Enzymes: EecoRI and SpeI (1uL each)
 * Total volume: RFP chlor backbone (bb): 14.50 + ccd chlor bb: 14.50
 * Add 30uL H2O to each digest after 1 hour

Gel verification on RD
Gel orientation: Machine conditions: 0.5x TBE buffer, 100V, 60min
 * Protocol: biobrick "digest" protocol
 * Changes: load 15uL into each well


 * RFP chlor: 0.4g
 * ccdb chlor: 0.5g
 * ccdb E/P: 0.8g

Ligations

 * Protocol: biobrick method
 * 1) His + ccdB -> HC
 * 2) His + RFP -> HR
 * 3) no his + ccdb -> NHC
 * 4) no his + RFP -> NHR

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


 * gel extraction failed. No ligations conducted.