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The Molecular Biology Labs - LAB 5
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The Molecular Biology Labs

LAB 5: Preparing Overnight Bacterial Culture

| MATERIALS | SUPPLIES | PROCEDURES | RESULTS |

Materials:

  1. Sterile LB medium (Luria-Bertani Medium) with or without antibiotic:Teacher's note: The presence of antibiotic depending the presence of antibiotic resistance on the plasmid DNA transformed into the bacteria. E coli DH5 cells should not be used with any antibiotic. E coli DH5 cells with pBC should require chloramphenicol. E coli DH5 cells with pGFP should require ampicillin.
    • water 500 ml
    • bacto-tryptone 10 g
    • bacto-yeast extracts 5 g
    • sodium chloride 10 g
    • stir to dissolve
    • pH to 7.0
    • autoclave to sterilize
    • cool to 50-60 degrees C
    • add antibiotic if neededTeacher's note: Final concentration of chloramphenicol is 100 ug/ul. Stock solution of chloramphenicol can be made in ethanol at a concentraion of 25 mg/ml and stored at -20 degrees C.
  2. Agar plate with bacterial colonies:
    • scrape bacteria glycerol stock such as E coli DH5 (LifeTechnologies) or scratch a single colonies from an old agar plate with bacteria colonies that has been stored at 4 degrees C.
    • streak onto an agar plate with or without antibioticTeacher's note: The presence of antibiotic depending the presence of antibiotic resistance on the plasmid DNA transformed into the bacteria. E coli DH5 cells should not be used with any antibiotic. E coli DH5 cells with pGFP should require ampicillin. E coli DH5 cells with pBC should require chloramphenicol.
    • incubate the plate inverted overnight at 37 degrees C
Supplies:
  1. Shaker at 37 degrees C
 Procedures:
  1. Scratch a single colony from the agar plate with bacterial colonies using a sterile small micropipetter tip (use a flamed forceps to hold the tip).
  2. Drop the tip in 2.5 ml of LB with or without antibiotics and shake at about 200 rpm for 4-8 hours at 37 degrees C.
  3. Use 0.5 ml of this bacteria culture to inoculate 100 ml of LB with antibiotics.
  4. Shake at about 200 rpm overnight at 37 degrees C.
 Results:
  1. Culture turns from transparent to cloudy after overnight growth. The overnight culture can be stored at 4 degrees C for several days while preserving the plasmid DNA integrity.

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