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

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Entry title
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)