IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/30

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Biofilm Track

Melody

Biofilm Growth Protocol Day 4

  • 15 minute Crystal Violet Stain on plates 100824E+M
    • Left in biohazard cabinet to dry overnight

Biofilm Growth Protocol Day 4

  • Took initial OD550 readings of plates 100823E+M
100823E Data
26 C 0.1883 0.0861 0.3199 0.0845 0.1662 0.0855 0.2453 0.0855
27 D 0.2003 0.0876 0.2839 0.085 0.1865 0.086 0.239 0.0861
28 E 0.1889 0.0868 0.2287 0.0856 0.2134 0.0862 0.263 0.0861
  • Note: Red = control
100823M Data
26 C 0.3861 0.0982 0.23 0.1764 0.2201 0.0853 0.14 0.0852
27 D 0.199 0.0882 0.243 0.0847 0.1975 0.0851 0.1933 0.0923
28 E 0.2243 0.0871 0.25890 0.0842 0.1662 0.0841 0.2676 0.0844
  • Note: Red = control


  • Took resolubilized OD550 readings on plates 100823ERS+MRS

10823ERS Data

26 C 0.1979 0.0798 0.2322 0.0806 0.2565 0.0923 0.30830 0.0867
27 D 0.2597 0.0827 0.3058 0.089 0.2793 0.0738 0.2879 0.0857
28 E 0.2679 0.0798 0.2712 0.0831 0.2719 0.0823 0.2556 0.087
  • Note: Red = control

100823MRS

25 <> 3 4 5 6 7 8 9 10
26 C 0.241 0.1677 0.25460 0.27470 0.2339 0.0862 0.2041 0.09
27 D 0.2581 0.0946 0.2673 0.0821 0.2444 0.0827 0.2604 0.2097
28 E 0.28870 0.1002 0.2796 0.0837 0.2243 0.0852 0.2705 0.0909
  • Note: Red = control


Biofilm Growth Protocol Day 1

  • O/N cultures of RN4220 and 8325-4 from TSA plates
  • Incubated @ 5:48 p.m.
  • To be inoculated into plates 100830M120 + 100830M222

QS

  • Colony PCR for agrCA from NCTC 8325

PCR Master mix Reagent 1x rxn volume (uL) Master Mix 5x rxn buffer 5 x15 75 10mM dNTP 0.5 x15 7.5 sdH2O 12.15 x15 182.25 Phusion polymerase 0.1 x15 1.5 MgCl2 2 x15 30 DMSO - 5% 1.25 x15 18.75 10uM fw primer (G1004) 2 x15 30 10uM re primer (G1005) 2 x15 30 Total 25 375


PCR Cycles:

   * 98C @ 3min
   * Cycle 27x:
         o 98C @ 10 sec
         o 72C @ 30 sec
         o 72C @ 40 sec
         o 72C @ 10 min 
   * 10C @ hold 
   * Ran gel for 1 hour. 80 V 0.5X TBE 1.3% agaorse. Two small 8-lane gel boxes. Also included 1 kb NEB ladders.
   * Results show many bands. Apparent weak bank/smudge of DNA above 2 kb region. Probably means many different plasmids present. The film probably has low antibiotic concentration.
   * Therefore, streaked some bacteria from each of the agrAC plates in the hopes of finding single colonies. Incubate at 37 °C.
   * Made DH5alpha O/N for competent cell making. Unfortunately, had to use Finlay lab incubator on the 3rd floor, which runs at 37 °C and 200 rpm.