DNA Extraction from Sterivex Filters using the
(Method provided by
Linda Amarel Zettler)
Autoclaved 2 mL microfuge tubes
Puregene kit (Puregene cat no.
D-7000A) from Gentra
Lytic enzyme (Puregene cat no.
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
Velcro strips or wire strips to
attach filters to the carousel in the hybridization oven
Hybridization oven with rotating
carousel (37-650C) or equivalent incubator with agitation.
- Take samples out of dewer and
let them thaw (~20min).
- Add 10 ul Lytic Enzyme.
- Incubate @ 37C for 30min on
rotating carousel (change to 65C when filters are removed).
- Add 10 ul Proteinase K from
- Vortex @ max speed for 10
- Incubate @ 65C for 1hr.
- 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.
- Subdivide this volume into 3
tubes (A,B,C) @~700ul per each
- Cool to RT ~20 min
- Add 233ul (per 700ul) of
Protein Precipitation Solution (from kit) to each tube.
- Vortex 20 sec to mix ppt
solution with cell lysate.
- Incubate on ice 15min.
- Centrifuge at max speed 5min.
- Pipette supernatant (from
protein pellet) into tube with 1 volume 100%IPA.
- Invert 50 times to mix.
- Centrifuge 5min @max speed.
- 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.
- 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.
- 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.