Difference between revisions of "Team:ICT-Mumbai/Experiments"

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       <a>Will we ever discover aliens?</a>
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       <a>Extraction of Exudates</a>
 
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         <p>GeneAll ExpinTM protocol used. Link provided below for the same:<br>
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https://www.cambio.co.uk/library/images/html_images/GeneAll/2013_Expin_protocol_web.pdf
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<li>Seeds of 4 crops were obtained as specimen.
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<li>Rice
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<li>Soybean
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<li>Tomato
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<li>Wheat
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<li>The seeds were sterilised by rinsing them with Ethanol a few times.
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<li>Common garden soil was collected, and sterilised in autoclave to avoid any outside contamination while collection of exudates.
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<li>Glass jars were filled upto quarter the volume with sterilised soil.
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<li>Sterilised seeds were sown in the jars, (4 jars per crop) and the crops were grown in clean room for 3 weeks.  
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<li>When the height of crops was sufficient after 3 weeks, the crops were uprooted carefully and soil was mixed with GC (Gas Chromatography) grade water.  
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<li>After incubating the mixture for 30 minutes, to allow all the exudates to be dissolved in water, the solution was filtered.
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<li>Filtered solution was evaporated in RotaVap. Vacuum was maintained to allow the evaporation at lower temperature as some of the exudates might be temperature sensitive.
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Revision as of 15:17, 2 October 2018

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Protocols

Bacillus subtilis transformation protocol

Components of media

SpC Medium

Add the following to 10ml of TBase

  • 100 ul of 50% glucose
  • 150 ul of 1.2% MgSO4.3H2O solution
  • 200 ul of 10% yeast extract
  • 250 ul of 1% CAS amino acids
SpII medium

Add the following to 10ml of TBase

  • 100 ul of 50% glucose
  • 700 ul of 1.2% MgSO4.3H2O solution
  • 100 ul of 10% yeast extract
  • 100 ul of 1% CAS amino acids
  • 50 ul of 0. 1M CaCl2
TBase
  • Dissolve the following in 1L of water and autoclave
  • (NH4)2SO4 - 2g
  • K2HPO4.3H2O - 18.3g
  • KH2PO4 - 6g
  • Trisodium citrate.2H2O - 1g

Procedure

  1. Streak a large patch of Bacillus subtilis cells onto an LB agar plate (2.5% LB and 1% agar)
  2. Incubate at 37 deg C overnight
  3. Scrape cell’s off the patch into 10 ml of freshly made SpC medium (for contents of SpC medium see below) till OD of tube becomes approximately 0.5
  4. Incubate at 37 deg C and 220 rpm for 5 hours
  5. Inoculate 200ul of this culture into 10 ml of freshly prepared SpII medium (for contents of SpII medium see below)
  6. Incubate at 37 deg C and 170 rpm for 90 min
  7. Pellet cells in 1.5 ml microcentrifuge tubes. Decent and store supernatant
  8. Resuspend pellets in 200ul of supernatant
  9. Add 15ul of plasmid (pBS1C backbone)
  10. Incubate at 37 deg C
  11. Plate on LB agar plates (2.5% LB and 1% agar) containing chloramphenicol (25 ug/ml)
  12. Incubate overnight at 37 deg C to grow colonies

Plasmid Preparation

GeneAll ExprepTM plasmid SV mini protocol used. Link provided below for the same:
GeneAll Prep

Extraction of Exudates

GeneAll ExpinTM protocol used. Link provided below for the same:
https://www.cambio.co.uk/library/images/html_images/GeneAll/2013_Expin_protocol_web.pdf

How much does the Earth weigh?
  1. Seeds of 4 crops were obtained as specimen.
    1. Rice
    2. Soybean
    3. Tomato
    4. Wheat
  2. The seeds were sterilised by rinsing them with Ethanol a few times.
  3. Common garden soil was collected, and sterilised in autoclave to avoid any outside contamination while collection of exudates.
  4. Glass jars were filled upto quarter the volume with sterilised soil.
  5. Sterilised seeds were sown in the jars, (4 jars per crop) and the crops were grown in clean room for 3 weeks.
  6. When the height of crops was sufficient after 3 weeks, the crops were uprooted carefully and soil was mixed with GC (Gas Chromatography) grade water.
  7. After incubating the mixture for 30 minutes, to allow all the exudates to be dissolved in water, the solution was filtered.
  8. Filtered solution was evaporated in RotaVap. Vacuum was maintained to allow the evaporation at lower temperature as some of the exudates might be temperature sensitive.
How do airplanes stay up?

Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Elementum sagittis vitae et leo duis ut. Ut tortor pretium viverra suspendisse potenti.