IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/18

iGEM Project name 1

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Biofilm Track Eric F. + Melody Misc. Streaked TSA plates of 8325-4 and RN4220, previous ones did not have enough individual colonies to be useful Autoclaved pipette tips Prepared 0.1% + 0.01% Crystal Violet (by accident) - was of no use Biofilm Growth Protocol Day 5+ - 100811a + 100811b Resolubilized 100811a + b with 95% ehtanol Corresponding plates were labelled 100811a-b and 100811b-b Placed in biosafety cabinet, waiting for plate reader to be free Took readings Biofilm Growth Protocol Day 3 - 100816M + 100816E Followed day 3 protocol Biofilm Growth Protocol Day 4 - 100814M + 100814E Stained with (what we later realized to be) 0.01% Crystal Violet Took readings, did NOT resolubilize today Readings Took readings on 100811a-b, 100811b-b, 100814M and 100814E All readings were garbage will place below soon Decided to ethanol lid and bottom of plates before re-doing measurements 100811a-b Readings 25 <> 3 4 5 6 7 8 9 10 26 C 0.0792 0.0845 0.0855 0.0807 0.0814 0.0777 0.0828 0.0891 27 D 0.0773 0.0789 0.0823 0.0829 0.0805 0.08 0.081 0.0819 28 E 0.0803 0.0812 0.0813 0.0846 0.0788 0.084 0.0827 0.085 29 F 0.0778 0.0861 0.0796 0.0849 0.0834 0.0812 0.0841 0.0796 Red = control 100811b-b Readings 24 <> 1 2 3 4 5 6 7 8 9 10 11 12 25 A 0.0895 0.081 0.086 0.0854 0.074 0.075 0.0775 0.0763 0.077 0.0745 0.0874 0.0932 26 B 0.081 0.0871 0.0825 0.0859 0.0785 0.081 0.0866 0.0805 0.0793 0.0854 0.1023 0.0841 27 C 0.0823 0.0786 0.0827 0.0847 0.076 0.0803 0.0824 0.0791 0.0775 0.0841 0.0857 0.095 28 D 0.0818 0.0903 0.0783 0.0816 0.0821 0.0828 0.0812 0.0766 0.0812 0.0831 0.0787 0.0833 29 E 0.0901 0.0916 0.0799 0.0806 0.0816 0.0829 0.0822 0.0884 0.0866 0.0822 0.0824 0.0911 30 F 0.0823 0.0823 0.0839 0.0811 0.0821 0.0814 0.0818 0.0821 0.0839 0.0824 0.0849 0.0812 31 G 0.0781 0.0778 0.0804 0.0833 0.088 0.0794 0.0831 0.0905 0.0825 0.0869 0.083 0.0826 32 H 0.0987 0.0907 0.0902 0.0814 0.0814 0.0825 0.0844 0.0882 0.0792 0.0843 0.0821 0.0938 Red = control 100814M Readings 25 <> 3 4 5 6 7 8 9 10 26 C 0.0888 0.2545 0.1799 0.2674 0.1814 0.1559 0.1363 0.1061 27 D 0.2585 0.3001 0.299 0.1342 0.0908 0.1863 0.1828 0.1336 28 E 0.225 0.2138 0.2287 0.1308 0.0944 0.1467 0.1435 0.1338 29 F 0.0957 0.1552 0.1509 0.159 0.1202 0.1103 0.1079 0.0906 Red = control 100814E Readings 25 <> 3 4 5 6 7 8 9 10 26 C 0.0938 0.1238 0.1038 0.0931 0.1034 0.1422 0.109 0.0994 27 D 0.1042 0.164 0.2106 0.1101 0.1065 0.101 0.1104 0.124 28 E 0.1003 0.1712 0.1296 0.1099 0.1092 0.1079 0.1577 0.1189 29 F 0.1638 0.1444 0.148 0.0999 0.1926 0.1471 0.1397 0.1453 Red = control dspB Mini-prep 3T1,3T2,4T1,4T2 Protocol: Rafael's alkaline lysis Nanodrop [ng/uL]: 3T1: 2541.7 3T2: 3053.8 4T1: 3357.5 4T2: 3587.8 Sample Volume of sample (uL) Volume of H2O 3T1 1.574 8.426 3T2 1.309 8.691 4T1 1.191 8.809 4T2 1.115 8.885 Primers: VF2 and VR 5uM each; 6uL in total (require 1uL of 5uM primers) =QS Track Miniprepped agrCA (following Raf's alkaline lysis protocol) from distribution (after following cPCR program using UBC iGEM protocols) No colonies for agrCA (natural form) showed up yesterday (both agrCA by itself and agrCA with terminator) Therefore, PCR'd more agrCA. Used same program, amount of reagent, and protocol as August 11 agr CA PCR. Ran on gel with 1.25% agarose, 0.5X TBE, 40 minutes, 100 V Bands showed up where expected (~2.1 kb) Used Qiagen PCR purification kit to purify agr CA product. Checked DNA quality with nanodrop. A260/280=1.90. A260/230=1.70. [DNA]=68.7 ng/μL.