DNA Extraction from Sterivex Filters using the Puregene Kit

(Method provided by Linda Amarel Zettler)

Materials

Gloves, parafilm, Autoclaved 2 mL microfuge tubes

Puregene kit (Puregene cat no. D-7000A) from Gentra

Lytic enzyme (Puregene cat no. D-60K4)

Note: Lytic Enzyme is a lyticase similar to Zymolase that breaks the 1-3 beta glucose linkages in bacterial cell walls. It is suspended in a proprietary glycerol solution at a concentration of 4000 U/mL. Alternatively, Lysozyme (50mg/mL) made up in autoclaved water can be added at 67ul per sample.

Proteinase K (20 mg/ml) from the kit

Isopropanol 100%

Ethanol 70%

Velcro strips or wire strips to attach filters to the carousel in the hybridization oven

Equipment

Microcentrifuge

Vortex

Hybridization oven with rotating carousel (37-650C) or equivalent incubator with agitation.

Protocol

  1. Take samples out of dewer and let them thaw (~20min).
  2. Add 10 ul Lytic Enzyme.
  3. Incubate @ 37C for 30min on rotating carousel (change to 65C when filters are removed).
  4. Add 10 ul Proteinase K from refrigerator.
  5. Vortex @ max speed for 10 seconds.
  6. Incubate @ 65C for 1hr.
  7. Attach a 3 cc syringe to the top (female end) of sterivex filter and use the plunger to push the liquid into a sterile 2 ml labeled microfuge tube. If necessary attach and repeat to push full volume out.
  8. Subdivide this volume into 3 tubes (A,B,C) @~700ul per each
  9. Cool to RT ~20 min
  10. Add 233ul (per 700ul) of Protein Precipitation Solution (from kit) to each tube.
  11. Vortex 20 sec to mix ppt solution with cell lysate.
  12. Incubate on ice 15min.
  13. Centrifuge at max speed 5min.
  14. Pipette supernatant (from protein pellet) into tube with 1 volume 100%IPA.
  15. Invert 50 times to mix.
  16. Centrifuge 5min @max speed.
  17. Pour off supernatant and drain on a paper towel. Add 700 ul of 70% ethanol and invert the tube several times to wash the DNA pellet.
  18. Centrifuge at maximum speed for 5 minutes. Pour off supernatant. Invert and drain the tube on a paper towel. Air dry for ~20 minutes until dry.
  19. Resuspend each pellet in 10 ul of hydration buffer provided with the kit. This will give a total of 30 ul of concentrated DNA per filter sample.