Gill:Rapid bacterial DNA prep

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Low-yield DNA suitable for PCR.

Materials

  • Sterile needles or toothpicks
  • Sterile microtubes
  • Lysis buffer (in ddH2O): 0.25% (w/v) SDS, 50 mM NaOH; do not autoclave, but store as frozen aliquots for long-term storage (>2 weeks).
  • Heat block at 95 ºC or boiling water bath (~100 ºC)
  • Autoclaved ddH2O or other molecular biology-grade water

Procedure

  1. Using a sterile toothpick or needle, pick a small amount of bacteria from a colony and deposit it in 20 µl of lysis buffer in a 1.5 ml microtube. The amount of bacteria should be a glob of about 1 mm in diameter. Tap the tube gently to suspend the bacteria evenly.
  2. Heat at 95 ºC for 15 min, or boil for 5 min. Centrifuge briefly to collect the liquid to the bottom of the tube.
  3. Add 180 µl of sterile ddH2O and mix.
  4. Centrifuge again at high speed in a microfuge (14000 - 16000 x g) for 5 min.
  5. Transfer 50 µl of the supernatant to a new tube, store frozen. Use 1 - 2 µl of this template per 50 µl PCR reaction.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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