User:David Johnston Monje/Notebook/Maize Endophyte Biofertilizers/2011/10/24

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The Daily Grind

  • Read a daily paper: Great topic about plant invasion being influenced by soil microbes - / Here's a good review about fungal TRFLP -
  • Do EF1 and PR1 directed PCRs directly on RNA samples to check for DNA contamination.
  • Once Derek replies, order Max550 labelled 1392R for PCR with 799F.
  • Photograph plates of bacteria - do TRFLP of soil DNA in triplicate per sample(sand, Mex, Can)
  • Apparently we got healthy and diseased strawberry rhizosphere soil, which Amy suggests I use for defining strawberry rhizosphere.
  • Organize/plan strawberry soil experiment (also known as California soil), brainstorm Trevor Charles metagenomics, plus other
    • Appears to me that we should simply do TRFLP on these samples for bacteria and fungi. Fungal primers I should use appear to include ITS1F and ITS4 (Use of the ITS primers, ITS1F and ITS4, to characterize fungal abundance and diversity in mixed-template samples by qPCR and length heterogeneity analysis - Another paper develops a pair of nested primers for amplification of fungal DNA to the exclusion of Oomycetes ( We may want to get specific primers for Oomycetes too, as these aforementioned primers appear to miss them.
    • For TRFLP, it appears that ethanol precipiation might be a viable technique, "Digests were desalted by precipitation with 96–100% (v/v) ethanol and 0.25 mol l) 1 sodium acetate, and left to precipitate overnight at 20 degrees C. Tubes were then centrifuged at 14 000 g at 4 degrees C for 15 min, washed with 70% (v/v) ethanol and resuspended in nuclease-free water, and aliquots (1 ul) were mixed with 38.75 umol per L of Beckman Coulter sample loading buffer and 0.25 ul of Beckman Coulter size standard 600 (High Wycombe, Bucks, UK)." from this publication -