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

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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

 * Note: Red = control

100823M Data

 * Note: Red = control


 * Took resolubilized OD550 readings on plates 100823ERS+MRS

10823ERS Data

 * Note: Red = control

100823MRS

 * 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
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
 * Colony PCR for agrCA from NCTC 8325

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.


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