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

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Autoclaved Pipette Tips

 * HUNDREDS OF THEM!

Made Ethanol

 * Like a boss

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

 * Note: Red = control

100816M aka Eric's Ethanol Shame Plate Data

 * 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

 * 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 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.


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