Gill:Rapid bacterial DNA prep
Revision as of 12:03, 17 January 2014 by Jacqueline K. Grimm (New page: 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 fro...)
Low-yield DNA suitable for PCR.
- 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
- 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.
- Heat at 95 ºC for 15 min, or boil for 5 min. Centrifuge briefly to collect the liquid to the bottom of the tube.
- Add 180 µl of sterile ddH2O and mix.
- Centrifuge again at high speed in a microfuge (14000 - 16000 x g) for 5 min.
- Transfer 50 µl of the supernatant to a new tube, store frozen. Use 1 - 2 µl of this template per 50 µl PCR reaction.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!