Team:ICT-Mumbai/Experiments

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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

Gel Purification and PCR Purification

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

Extraction of Exudates
Procedure
  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.
Preparation of E coli DH5α competent cells
  1. Inoculate DH5 alpha cells - 10 ul from the glycerol stock into LB medium (2.5%).
  2. Incubate overnight at 37 deg C.
  3. Next day, subculture thi culture into fresh tubes containing 10ml LB medium (2.5%) each -100 ul of the overnight culture added to the LB media.
  4. Incubate at 37 deg. C for approximately 2-3 hrs till you get an OD of around 0.6
  5. Place these LB tubes and 0.1 M CaCl2 on ice (all further steps to be carried out on ice in a LAF)
  6. Distribute each tube containing 10 ml culture into two / four microcentrifuge tubes.
  7. Centrifuge the tubes and discard the supernatant. Ensure complete removal of the supernatant.
  8. Resuspend pellets in 1 mL of 0.1M CaCl2.
  9. Keep them in the ice for half an hour.
  10. Now centrifuge the tubes again and discard the supernatant.
  11. Resuspend pellets in 200 ul of 0.1 M CaCl2.
  12. Once suspended add 40 ul of the glycerol.
  13. Distribute this 240 ul of solution in each microcentrifuge tube into two microcentrifuge tubes with 120 ul in each.
  14. Now store these cells in the -80 deg C.
Transformation into E coli DH5α
  1. The competent cells are removed from -80 deg C and thawed on ice
  2. 1-2 ul of plasmid is added to 100 ul of competent cells (i.e. into one microcentrifuge tube containing competent cells – the competent cells are made and stored in a way that each microcentrifuge tube has 100 ul)
  3. The cells are kept on ice for 30 min
  4. The cells are then heat shocked by placing them in a water bath at 42 deg C for 90 seconds
  5. The cells are further allowed to rest on ice for 5 min
  6. 700 ul of autoclaved LB media (2.5%) is added to each microcentrifuge tube
  7. Microcentrifuge tubes are now incubated at 37 deg C for a period of time depending upon the antibiotic resistance provided by the plasmid:
    • Chloramphenicol resistance – 2 hours
    • Ampicillin resistance – 1 hour
    • Kanamycin resistance – 1 and a half hour
  8. The cells are now plated onto LB + agar plates (2.5% LB and 1% agar) having the following concentration of the required antibiotic:
    • For Chloramphenicol – 25 ug/ml
    • For Ampicillin – 100 ug/ml
    • For Kanamycin – 100 ug/ml
  9. Plates are incubated at 37 deg C overnight for colonies to grow.
Preparation of E coli DH5α competent cells
  1. Inoculate DH5 alpha cells - 10 ul from the glycerol stock into LB medium (2.5%).
  2. Incubate overnight at 37 deg C.
  3. Next day, subculture thi culture into fresh tubes containing 10ml LB medium (2.5%) each -100 ul of the overnight culture added to the LB media.
  4. Incubate at 37 deg. C for approximately 2-3 hrs till you get an OD of around 0.6
  5. Place these LB tubes and 0.1 M CaCl2 on ice (all further steps to be carried out on ice in a LAF)
  6. Distribute each tube containing 10 ml culture into two / four microcentrifuge tubes.
  7. Centrifuge the tubes and discard the supernatant. Ensure complete removal of the supernatant.
  8. Resuspend pellets in 1 mL of 0.1M CaCl2.
  9. Keep them in the ice for half an hour.
  10. Now centrifuge the tubes again and discard the supernatant.
  11. Resuspend pellets in 200 ul of 0.1 M CaCl2.
  12. Once suspended add 40 ul of the glycerol.
  13. Distribute this 240 ul of solution in each microcentrifuge tube into two microcentrifuge tubes with 120 ul in each.
  14. Now store these cells in the -80 deg C.
Preparation of E coli DH5α competent cells
  1. 1% agarose gels are cast:
    • Each casting unit can hold 30 ml of melted gel.
    • Appropriate amounts of agarose powder are weighed out (for 1% W/V gels) into a flask
    • This powder is added to 0.5X TBE buffer and heated till the powder dissolves
    • EtBr dye is added to the flask (2 ul for every 30 ml of gel)
    • Combs are placed onto the casting units and the flask is emptied into the casting untis
    • Gels are allowed to cool down and solidify
  2. Solidified gels are placed in an electrophoresis tank containing 0.5X TBE buffer
  3. Samples are loaded into each well
  4. A marker is loaded into one of the gels
  5. The electrodes are attached and potential difference provided to run the gel
  6. After gel has run, the bands are viewed under UV light