Maheshri:TransformationB

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Bacterial transformation

Reagents

  • TB (Terrific broth without antibiotics)
  • Competent bacteria (stored at –80C)
  • TB/Amp plates

Protocol

  1. Take an aliquot of chemically competent bacteria from the –80C freezer and thaw on ice. Bacteria must be kept cold at all times until the heat shock, or they will lose competency.
  2. Add diluted plasmid DNA stock (1-10 ng, e.g. dilute 1 uL of 1 ug/uL plasmid stock into 1 mL of water, and add 1 uL to bacteria) or 10 uL ligation reaction to the bacteria. Do not leave bacteria thawing for longer that 15 minutes. Also, be sure that the volume of DNA solution added to the bacteria doesn’t exceed 20% of the bacterial volume.
  3. Incubate bacteria on ice for 30 minutes.
  4. Heat shock by placing in 42C water bath for 30 seconds.
  5. Place tube on ice, and add 0.5 mL of TB (without antibiotics).
  6. Shake for 30 minutes at 37C (to allow the bacteria to begin expressing antibiotic resistance).
  7. Plate on an LB/antibiotic plate (most commonly ampicillin).

Note: Plates can be placed at 37C prior to the transformation to allow them to dry slightly.

Support protocol: preparation of antibiotic agar plates

You will need:

  • TB
  • Agar
  • 10 cm plastic Petri dishes (e.g. Falcon)
  • Antibiotic stock (most commonly ampicillin or kanamycin):
100 mg/mL	ampicillin (1000x, in water, store at –20C)
50 mg/mL	kanamycin (1000x, in water, store at –20C)

Make TB media, and add agar at 2% (w/v). Autoclave, and allow to cool until comfortable to hold with bare hands (~50C, very important since ampicillin degrades at higher temperatures). Add antibiotic to the proper concentration, and pour plates using proper sterile technique. Store plates at 4C.

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