Sam:LabNotes/Microbiome/2009-2-17

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Revision as of 19:04, 8 March 2009 by >Sam Chiang (New page: ='''LB Agar plates Preparation & E. coli Bacteria Enrichment'''= ==Objective== *Preparing LB agar plate for constructing standard curve of OD600 vs CFU/mL. *Enrich the E. coli bacteria ...)
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LB Agar plates Preparation & E. coli Bacteria Enrichment[edit]

Objective[edit]

  • Preparing LB agar plate for constructing standard curve of OD600 vs CFU/mL.
  • Enrich the E. coli bacteria for further application.



LB agar plate preparing[edit]

Materials[edit]

  • LB Miller agar powder (EMD chemical Inc.)
  • 1 L Flask
  • Petri dish -- 20 plates / 500mL agar
  • Antibiotics - Kanamycin [1](50mg / mL, 1000X, stored in -20 degree)


Procedures[edit]

  • Weigh the LB agar powder 37 g / 1 L -> 18.5 g / 500 mL. Dissolve 18.5 g LB agarose mix into 500 mL diH2O in a 1 L-flask. Makes two of these mixture.
    • To stir the mixture for dissolving is not necessary. The autoclaving will help dissolving the mixture by itself.


  • Wrap the flask cap with foil and tape the autoclave-indicator tape.


  • Put the flasks on autoclave tray and autoclave the flask using the machine on 3rd floor.
    • Sign the log (name, date, which cycle been used, and machine condition)
    • Open the door by press "open".
    • Take out the upper iron tray if necessary. Put our mixture on the lower iron tray. Press 'close' to close the door.
    • Press '6' for selecting liquid cycle (121 degree, 30 min). Press 'start'. The total autoclaving time is about 45 min.
    • When the procedure is done. Press 'open' to open the gate. Wear gloves and take our media out.


  • Gently stirring the media for better mixing. Cool the Agar media in RT for 30 min or until it become 60 degree. At the same time thaw the antibiotics on ice.


  • Add 500 uL of antibiotics (1000X) into the flask (containing 500 mL agar media). Gently stirring the media for mixing.


  • Set up the gas-flaming. Pour the agar plate beside the flaming set.


  • Cool down the finished plates for 1 hr beside the flame.


  • Store the solid plates in sleeve in 4 degree.



Bacteria enrichment[edit]

Materials[edit]

  • Pre-made sterile LB broth (which brand?)
  • Antibiotics - Kanamycin (50 mg / mL, 1000X, stored in -20 degree)
  • 15-mL screw cap tubes (Corning brand)
  • 250-mL screw cap bottle (one-time use, Corning brand)


Procedures[edit]

  • Thaw the E. coli cells on ice


  • Prepare the LB-broth w/-antibiocs Kanamycin
    • Add 250 uL Kanamycin into 250 mL LB broth in a 250-mL bottle. Mix the mixture by shaking gently.


  • Preparing the following preps
 Tube#        Media                 Innoculatin
 -----------------------------------------------
 #1     LB w/-kanamycin, 5 mL     E.coli 5.0 uL
 #2     LB w/-kanamycin, 5 mL     E.coli 2.5 uL
 #3     LB w/o-kanamycin, 5 mL    E.coli 5.0 uL
 #4     LB w/o-kanamycin, 5 mL    E.coli 2.5 uL
  • Mix with gentle vortexing. Lose the cap for better air circulation. Incubate in the 37 degree water bath overnight.
    • Incubation start at 5:30 PM


****************Next Day*****************


Results (check on 9:40 AM)[edit]

  • The #1 and #2 didn't grow, but both #3 and #4 (w/o antibiotics) grew very well.
  • Innoculation with differnt volumes (2.5 uL or 5.0 uL) makes no difference.

Discussion[edit]

  • It is possible the cells taken from -80 freezer weren't adapted to the most optimal condition for culturing yet.
    • We could try to pass some grown culture (from w/o-antibiotics media) on with-antibiotics media and culture them again.
  • The concentration of Kanamycin added in LB broth is too strong?
    • Tansformed E. coli can resist to Kanamycin around 50 mg~100 mg / L (agar or broth). The stock solution we used is 50 mg/mL, which is the 1000X of the recommanded conc. => The dosage we added in broth and agar plates is correct.


Update (02-18-09)[edit]

  • It is probably the E. coli we used from TOPO clone (Invitrogen) were wild-type Bacteria, which don't have resistent gene against to Kanamycin.