User:David Johnston Monje/Notebook/Maize Endophyte Biofertilizers/2012/11/16

Applied Soil Microbial Ecology

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Remains of the Day Extracting DNA from roots and rhizospheres of the last tomtatoes today (sterile sand and 0.1% molasses) - after weighing and photographing, I'm going to surface sterilize the pooled roots and shoots, then grind, dilute with sodium phosphate buffer and plate them on R2A, PDA-C and some Verticilium media, looking for fungi This was done by sterilizing with 2% bleach for 10 min (2x), rinsing with water, 10 min with 95% ethanol and then rinsing 3 times with autoclaved water. Roots and shoots were ground in morter and pestle, 100 ul of "juice" that came out was diluted 10X into 900 ul of water, then again, and again, and the last 2 dilutions were plated (100 ul of each) on half a plate of the medias R2A, SPT, and PDA-C(holamphenicol?). Likewise, rhizosphere washes from both the sterile sand and 0.1% molasses treated plants were pooled (100 ul from each of the 5 plants per treatment) and diluted, with 100 ul of the 10,000X and 1,000X dilutions being plated. These rhizosphere washes were generated by adding 10 mL of deionized water to unwashed roots in a falcon tube, shaking, then putting the now dirty water into its own 15 ml falcon tube. Dilutions were done with autoclaved 1M sodium phosphate solution. Made glycerol stocks from each juice sample by putting 700 ul into 400 ul of 75% glycerol, then putting the cryovials (tops labelled as TVD) into the -80 freezer. Work on Terrabiogen report