FastDNA Spin Kit Protocol (MP Biomedical)
1)
2)
3)
4)
5)
Filter the water sample according to the Water Sample Filtration Protocol and add
the filter disc to the Lysing Matrix A tube (orange-cap tube).
Add 1000μL CLS-TC into the orange-cap tube.
Vortex the tube for 1 min @ max speed using the disruptor genie.
Centrifuge the orange-cap tube @ 14,000xg for 10minutes.
Transfer the supernatant (liquid portion) to a fresh, yellow, 2mL centrifuge tube.
Avoid transferring any of the beads from the orange-cap tube.
You should be able to recover a majority of the liquid, ~700-1000μL. Less
liquid recovery = less DNA extracted.
6)
Invert the binding matrix bottle to mix and re-suspend any precipitated matrix.
Add an equal volume of binding matrix to your lysate.
Ex: if you recover ~800μL of the lysate, add 800μL of binding matrix
7)
Incubate @ room temperature (on the bench top) for 5 minutes. Invert the tube
10 times to mix every minute.
Mixing the lysate and binding matrix during incubation increases binding
efficiency and therefore DNA recovery
8)
Transfer ½ the volume to a SPIN MODULE and centrifuge @ 14,000xg for 1
minute, then empty the catch tube (the liquid that has spun through).
The SPIN MODULE is made up of a FILTER (upper portion) and a CATCH
TUBE (lower portion).
FILTER
SPIN MODULE
CATCH TUBE
9) Add the remaining volume and repeat step 8.
10) Add 500uL SEWS-M and gently re-suspend the binding matrix pellet that has
formed on the FILTER. SLOWLY AND GENTLY pipet up and down (go to the first
stop on the pipet to avoid air bubbles) until the binding matrix pellet is resuspended.
11) Centrifuge @ 14,000xg for 1 minute, discard the flow through in the CATCH TUBE.
12) Centrifuge again @ 14,000xg for 2 minutes and discard the flow through.
13) Replace the CATCH TUBE with a new RECOVERY TUBE.
14) Elute the DNA by gently re-supsending the binding matrix pellet in 100uL DES
(just like re-suspension at step 10).
15) Cap the tube and incubate it @ 55°C for 5 minutes.
16) Centrifuge @ 14,000xg for 1 minute to elute the DNA. You can throw out the SPIN
MODULE (both the FILTER and the previously discarded CATCH TUBE).
17) Label your tube DNA and the date (and any other relevant information) and store
it @ -20°C.