User:David Johnston Monje/Notebook/Maize Endophyte Biofertilizers/2010/05/12

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  • Gonna do a functional trial of Arabad constructs in pDRIVE in JM109 cells. Will grow O/N of GFP, ACC deaminase, and Kp342 alsSD, TB1 RNAi, and PDS RNAi from glycerol stocks overnight in SOB media. Tommorow, will take pellets, wash them with 750 ul of sodium phosphate buffer 3 times, resuspend them in sodium phosphate buffer to OD600 = 1.0, and use 100 ul to inoculate test media.
    • For ACC, test media (antibiotic free) will be 2 mL N free M9 (plus 20 uL 1M glucose) with 100 ul of 0.5 M ACC and 200 ul of 10% Arabinose (controls will be A - 2 mL N free M9 with 100 ul of 0.5 M ACC (no arabinose) and B - 2 mL N free M9 200 ul of 10% Arabinose). lacZ::ACC deaminase from Glick will be used in all these treatments, with 10 ul of IPTG added per tube.
    • For all other constructs, 2 mL of SOC media (no antibiotics) with 200 ul of 10% Arabinose or without Arabinose will be innoculated with 100 ul of OD600 = 1.0 washed cells to inoculate test media. GFP tubes will be compared for GFP, alsSD tubes will be scored for red colour in Barrit's reaction, and RNAi constructs will be scored for OD and growth (does RNAi kill the cells?)



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