Chang Lab:Notebook/CBE/08/148/2008/12/04

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Preparation of LB Agar:

  • Weight 14g of Agar and dissolved in 400ml of distill water
  • Autoclaved the flask at 121 degree celsius for 15 minutes
    • (An autoclave is a pressurized device designed to heat aqueous solutions above their boiling point at normal atmospheric pressure to achieve sterilization)
  • As time is needed for the pressure to built up for the temperature to rise, the whole process took about 2 hours to complete
  • The temperature was let to cool below 75 degree celsuis before we could open the autoclave machine to take out our flask
  • The LB Agar was then pour into about 40 petri disks in the ventilation hood.
  • The LB Agar in the disk was cooled down before storing them in the fridge
  • The remaining LB Agar was stored in the 70 degree incubator

Preparation of LB Broth:

  • Weight 14g of LB Broth and dissolved in 400ml of distill water
  • The flask was autoclaved at 121 degree celsius for 15 minutes
  • Upon completion, the LB Broth is stored in the 70 degree incubator

Ecoli Culturing:

  • Before we start, the UV light in the Biosafety hood was switched on for about 20 minutes
  • After 20 minutes, turn off the UV and wait for about 5 minutes before using the safety hood.
    • (This is to allow all the O3 to clear)
  • Ecoli Strand (K12 W3110) was mixed with LB Broth in the ratio 1:9 in the 15ml test tube
  • The test tube is left in the incubator at 30 degree, 30rpm for 24hour

Plate Streaking:

  • After 24 hour, the Ecoli culture was taken out of the incubator into the biosafety hood
  • One loopful of E coli streaked (quadrant streaking method) onto LB(A) plate (as immediate culture)
  • A slant stock culture was also prepared with the same method
  • Two E coli plate and slant was incubated at 37 C overnight (time start: 1020 hrs)

Improtent Notes

  • Always Parafilm petri dishes when storing (this is to avoid contaimination)
  • Overturn dishes upon storing(this is to avoid the water droplets from falling onto the agar)
  • For streaking, we must always ensure that the agar is free from moisture before we streak)




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