User:David Johnston Monje/Notebook/Maize Endophyte Biofertilizers/2011/08/02

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The Daily Grind

  • Bring 1 genotype/treatment (so 5 pots at a time) to graminas lab. Cut into shoots/roots, weigh, then put into sterile 50 mL falcon tubes, put 40 mL of wash solution in, sonicate for 10 min (take care to ensure cut roots/stems are not submerged). Tissues were then removed (roots retained) centrifuged at 30,000 g for 15 min at 4oC, generating a pellet. The supernatant was removed, and the pellet resuspended in 4 mL of wash buffer, subdivided into two 2 mL eppendorf tubes, then frozen for later DNA extraction.
  • With washed root tissue, a surface sterilization procedure was used including 10 minutes of washing in 3% bleach, 10 minutes of washing in 3% bleach, rinsing in distilled water, followed by a 10 minute of washing in 99% ethanol. Surface sterilized roots will then be withdrawn, crushed in a washed/flamed mortar/pestle, then re-inserted into falcon tube for a similar extraction as above (after sonification, root tissue will be discarded).
  • Need 100 --> 50 mL falcon tubes, 200 --> 2 mL falcon tubes, 4 L of wash buffer
  • Extracting sand grown plants today. Shoots were cut just above the bottom leaf (senesing)while the roots remained buried. Roots were gently shaken loose of the sand, shaken more to remove excess soil, and the rotten seed removed. 40 ml of buffer (potasium phosphate pH 7 plus tween 20) was added, and the falcon tubes were sonicated in a Thornton T7 sonicator (110V) at full (setting #12). Roots were washed similarly to Soil Swap study. (2.5% bleach, two times washed for 10 minutes in sonicator, followed by similar 10 minute 70% EtOH sonication, then washed in buffer and ground with mortars in the same falcon tubes they were washed in - juice collected for DNA extraction.) Falcon tubes were then centrifuged for 10 minutes at 10,000 rpm and 4 degrees on an Eppendorf 5810/R centrifuge Biomass measurements as follows.

Soil Maize Genotype Plant # Tissue Fresh weight (g)
Sterile sand Lenha 1 Shoot 1.2
Sterile sand Lenha 1 Root 2.8
Sterile sand Lenha 2 Shoot 1.3
Sterile sand Lenha 2 Root 2.7
Sterile sand Lenha 3 Shoot 1.8
Sterile sand Lenha 3 Root 2.1
Sterile sand Lenha 4 Shoot 1.4
Sterile sand Lenha 4 Root 3.4
Sterile sand Lenha 5 Shoot 0.8
Sterile sand Lenha 5 Root 0.9
Sterile sand BRS 1030 6 Shoot 1.1
Sterile sand BRS 1030 6 Root 4.3
Sterile sand BRS 1030 7 Shoot 1.6
Sterile sand BRS 1030 7 Root 4.2
Sterile sand BRS 1030 8 Shoot 2.1
Sterile sand BRS 1030 8 Root 5.1
Sterile sand BRS 1030 9 Shoot 1.3
Sterile sand BRS 1030 9 Root 4.7
Sterile sand BRS 1030 10 Shoot 2.7
Sterile sand BRS 1030 10 Root 6.8