User:David Johnston Monje/Notebook/Maize Endophyte Biofertilizers/2009/06/18

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The Daily Grind

 * Using VP broth (Peptone from meat 7.0; D(+)glucose 5.0; sodium phosphate 5.0, pH 5) I cultured Kp342 with pDKS-GFPuv (with or without Kanamycin), DH5alpha with GFPuv (with or without Kanamycin), JM109 (without Kanamycin), and DH5alpha with two different copies of the alsD operon in pDSK-GFPuv (with kanamycin). After 48 hours, I added 0.5 mL Barrit Reagen A (0.5 g 1Napthol in 10 mL EtOH) and 0.5 mL of Barrit Reagent B (40% potatssium hydroxide) to 1 ml of the bacterial cultures and waited 30 minutes to record a reaction. Interestingly, all the E. coli were negative (with or without plasmids). HOWEVER, Kp342 is positive when grown without kanamycin: the Kp342 with pDSK-GFPuv grown under antibiotic selection did not produce acetion. I am going to redo the test on the E. coli but exclude kanamycin this time....
 * Planting coca seed in a Promix PGX (half of them) and other half in 1/3 topsoil, 1/3 medium vermiculite, 1/3 perlite (adjusted to pH 5 - crap I forgot and its actually pH 7!!! maybe have to redo - I unpotted them and poured glacial acetic acid into the mix til pH was 5 and repotted them : pH of the proMix appeared to be 6 but I didn't mess with it). Putting them in the 10 hour, 28 degree growth room in a covered contained, each in their own pot. Hopefully will find a way to suspend the pots so they don't sit on the floor.


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