IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/02

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Melody

 * Vicki Ma took out O/n cultures of RN4220 and 8325-4 from the non-shaking incubator @ 11:00 a.m.
 * No contamination in control
 * Took out TSA plates spread with RN4220 and 8325-4 from non-shaking incubator @ 11:00 a.m.


 * Took out plate 100831M24 @ 3:20 p.m.
 * Took out plate 100831M26 @ 4:30 p.m.
 * No contamination in any of the controls

Biofilm Protocol Day 3

 * 3 x 300ul PBS wash on plates 100831M24 + 100831M26
 * 100831M24 heat fixed @ 4:48 p.m.
 * 100831M26 heat fixed @ 5:00 p.m.

Biofilm Protocol Day 2

 * Innoculate plates 100901M28 + 100901M30.

100901M28 + 100901M30 Plate Layout

 * randomized controls


 * 100901M28 incubated @ 4:06 p.m.
 * 100901M30 incubated @ 4:15 p.m.

Biofilm Protocol Day 4

 * 0.1% Crystal Violet Stain on plates 100830M120 + 100830M222

Restriction Digest
Enzymes: Use EcoRI and SpeI (1uL each) DNA: RFP chloraphenicol backbone, and ccdB chlorraphenicol backbone

Gel verification on RD
Gel orientation: Machine conditions: 0.5x TBE buffer, 90V, 60min Results:
 * Protocol: biobrick "digest" protocol
 * Changes: load 20uL into each well; 10uL for ladder

Gel extraction
ccdB chlor gel weight: 0.149g RFP chlor gel weight: 0.0872g EP chlor gel weight: 0.0334g
 * Using Invitrogen PureLink Quick Gel Extraction Kit (protocol inside kit)

Nanodrop:
 * [ccdB chlor] = 54.0ng/uL (wavelength = 220nm)
 * [RFP chlor] = 2.0ng/uL (wavelength = 220nm)
 * [EP chlor] = 7.2ng/uL (wavelength = 230nm)

Ligation

 * Protocol: biobrick method (number indicates tube number)
 * 1) His + ccdB -> HC
 * 2) no his + ccdB -> NHC
 * 3) his + impure ccdb (not extracted) -> iHC
 * 4) no his + impure ccdB (not extracted) -> iNHC
 * 5) AC + EP chlor -> AC (for Phillip)
 * 6) AC + impure EP chlor (not extracted) -> iAC (for Phillip)

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

Insert length = 1200bp (DspB); vector length: 2900bp 2. 1uL vector: 0.341uL no his *2 = 2uL vector: 0.681uL no his 3. 2uL vector: 1.06uL his 4. 2uL vector: 0.681uL no his 5. insert mass: 21.6ng 6. Can't do ligation because not enough insert
 * 1) insert mass: 67.03ng
 * 1uL vector: 0.531uL his *2 = 2uL vector: 1.06uL his
 * 1uL vector: 1.08uL insert *4 = 4uL vector: 4.32uL AC
 * Put in ~2uL of AC because not enough in the tube


 * Changes:
 * Added 2uL of Ligase buffer
 * Total volume: 10uL
 * Incubate at room temperature for 15 minutes

Transformations

 * Protocol: SOP
 * Add 10uL of ligation mixes to HC, NHC, iHC, iNHC, AC
 * Add 2uL of psb1C3 (ccdB) to control
 * Spread plate with 50uL
 * Plates put in 37C incubator at 7:48pm


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