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

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Biofilm Track

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

Row 3 4 5 6 7 8 9 10
C R R C R C 8 8 C
D R C R R 8 C 8 8
E R R C R 8 8 8 8
  • 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

25 <> 3 4 5 6 7 8 9 10
26 C 0.088 0.1711 0.0871 0.1781 0.26520 0.0876 0.201 0.0899
27 D 0.1575 0.0877 0.159 0.178 0.0885 0.2086 0.2007 0.2098
28 E 0.1404 0.1722 0.0877 0.09 0.0911 0.1961 0.0878 0.1733
  • Note: Red = Control

100824M Data

25 <> 3 4 5 6 7 8 9 10
26 C 0.1 0.2297 0.1008 0.1596 0.2858 0.107 0.30880 0.1014
27 D 0.1701 0.1034 0.2243 0.2292 0.1074 0.2834 0.2811 0.2646
28 E 0.2613 0.28 0.129 0.1379 0.1135 0.32410 0.1013 0.2958
  • Note: Red = Control


  • Resolubilized in 95% ethanol

100824ERS

</tr
25 <> 3 4 5 6 7 8 9 10
26 C 0.0858 0.29070 0.0824 0.2807 0.31060 0.0869 0.2732 0.0899
27 D 0.2961 0.0886 0.2548 0.2782 0.0802 0.32320 0.36630 0.2829
28 E 0.25890 0.3078 0.1923 0.2443 0.0957 0.333 0.0831 0.3118
  • Note: Red = Control

100824MRS

25 <> 3 4 5 6 7 8 9 10
26 C 0.104 0.2175 0.1014 0.2176 0.2177 0.1105 0.1784 0.1171
27 D 0.1705 0.0967 0.1966 0.2031 0.1184 0.2355 0.2417 0.2489
28 E 0.2067 0.226 0.1062 0.1086 0.0997 0.2381 0.1079 0.1886
  • 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

dspB Track

Restriction Digest

Restriction Digest Supermix
REAGENTS1 RXN VOLUME (uL)
Buffer 25
BSA0.5
  • 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

  • Protocol: biobrick "digest" protocol
  • Changes: load 15uL into each well

Gel orientation:

Gel orientation
ccdb chlorbbccdb chlorbbccdb chlorbb100bp ladderRFP chlorbbRFP chlorbbRFP chlorbb100bp ladderEP chlorEP chlorEP chlor

Machine conditions: 0.5x TBE buffer, 100V, 60min

  • 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.




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