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

iGEM Project name 1

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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 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 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.