IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/26

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Misc.

Melody + Eric F.

Autoclaved Pipette Tips

  • HUNDREDS OF THEM!

Made Ethanol

  • Like a boss

Biofilm Track

Eric F. and Melody

Biofilm Growth Protocol Day 5+ (100814 E + M aka Colourful Contaminate + Round Bottomed Failure)

  • Performed resolubilization
    • Used 150uL 95% ethanol instead of 200uL
    • The Round Bottomed Failure had more 0.1% crystal violet added
    • Crystal Violet was allowed to resolubilize for too long (30 minutes +)
  • No readings were taken - all information was scrapped
  • Plates discarded

Biofilm Growth Protocol Day 5+ (100816 E + M aka Shades of Purple and Eric's Ethanol Shame)

  • Performed first day 4 plate reading
100816E aka Shades of Purple Plate Data
25 <> 3 4 5 6 7 8 9 10
26 C 0.0858 0.1938 0.2189 0.27560 0.2284 0.219 0.2383 0.1244
27 D 0.3192 0.1921 0.1457 0.0866 0.0872 0.1915 0.2028 0.2058
28 E 0.0869 0.2389 0.17 0.1957 0.2133 0.2175 0.2499 0.2143
  • Note: Red = control
100816M aka Eric's Ethanol Shame Plate Data
25 <> 3 4 5 6 7 8 9 10
26 C 0.0862 0.34240 0.3238 0.3474 0.26140 0.1814 0.36860 0.3825
27 D 0.3526 0.3208 0.2759 0.1913 0.1912 0.30270 0.27130 0.25640
28 E 0.0873 0.3024 0.2958 0.269 0.3367 0.32590 0.3073 0.0866
  • Note: Red = control



  • Performed resolubilization
    • Used 150uL 95% ethanol instead of 200uL
    • Switched aliquots of 95% ethanol during Shades of Purple, distinct colour change noticed
  • Readings were taken
100816ERS aka Shades of Purple Redux Plate Data
25 <> 3 4 5 6 7 8 9 10
26 C 0.0821 0.0942 0.0966 0.0917 0.0997 0.1018 0.1014 0.1052
27 D 0.1013 0.0982 0.1009 0.0842 0.083 0.0927 0.0936 0.0967
28 E 0.082 0.0868 0.0867 0.0929 0.0844 0.266 0.2592 0.2127
  • Note: Red = Control


  • Only useful information was confirming the correct orientation of plates in the reader
  • Plates discarded

Biofilm Growth Protocol Day 4 (100823 E + M aka Eric's Pride 1 and Melody's Shame 1)

  • Stained with 0.1% crystal violet, left to dry overnight in biosafety cabinet

Biofilm Growth Protocol Day 3 (100824 E + M aka Eric's Pride 2 and Melody's Shame 2)

  • Performed washings with multi-channel pippeteman using unautoclaved pippete tips
  • Left overnight in biosafety cabinet

QS Track

  • A film of bacteria grew again on chloramphenicol plates.
  • Do not know why.
  • Picked colonies from agrAC (natural) + J23100, agrAC (natural) + B0014, agrAC (natural), and agr AC (distribution) + J23100.
  • Very difficult to do. Probably obtained many colonies at the same time.

Colony PCR

PCR Master mix
Reagent1x rxn volume (uL)Master Mix
5x rxn buffer5x1575
10mM dNTP0.5x157.5
sdH2O12.15x15182.25
Phusion polymerase0.1x151.5
MgCl22x1530
DMSO - 5%1.25x1518.75
10uM fw primer (G1004)2x1530
10uM re primer (G1005)2x1530
Total25375

PCR Tubes: 1,4,5,8,11,14,15,his,n1,n2,n3,n4,n5,W(H2O control)

  • Pick colony from respective plate and place in respective tubes

PCR Cycles:

  • 98C @ 3min
  • Cycle 27x:
    • 98C @ 10 sec
    • 72C @ 30 sec
    • 72C @ 40 sec
    • 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.