TITLE: MP Biomedicals FastDNA™ SPIN Kit
AUTHORS: Karen Keegan

[DESCRIPTION]
MP Biomedicals FastDNA‱
SPIN Kit for feces

[STEPS]
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
1. In a 2 mL Lysing Matrix E tube, add 500 mg feces sample, 825 μL Sodium
Phosphate Buffer, and 275 μL of PLS solution. Shake to mix. Vortex 10-15
seconds. 
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
2. Centrifuge samples at 14,000 x g for 5 minutes and decant supernatant.
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
3. Add 978 μL Sodium Phosphate Buffer and 122 μL MT Buffer.
Shake vigorously or vortex briefly to mix. 
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
4. Homogenize samples in the FastPrep 24 instrument at setting
6.0m/s for 40 seconds.
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
5. Centrifuge samples at 14,000 x g for 15
minutes. NOTE: Extending centrifugation to 15 minutes (did 15 instead of
5) can enhance elimination of excessive debris from large samples, or from cells
with complex walls.
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
6. Transfer the supernatant to a clean 2.0 mL centrifuge tube.
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
7. Add 250 μL of PPS solution, shake vigorously to mix, and
incubate at 4°C for 10 minutes. Do not vortex! Centrifuge samples at 14,000 x g
for 2 minutes.
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
8. While samples are centrifuging, add 1 mL of Binding Matrix Solution to a clean 15 mL
conical tube (not supplied).
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
9. Transfer supernatant to the Binding Matrix Solution in the
15 mL conical. Shake gently by hand to mix, then place on a shaker/rocker for
3-5 minutes.
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
10. Centrifuge samples at 14,000 x g for 2 minutes. Decant the
supernatant
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
11. Wash the binding mixture pellet by gently resuspending with
1 mL Wash Buffer #1.
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
12. The following step will require two spins. First, transfer
approximately 600 μL of the binding mixture to a SPIN Filter tube and
centrifuge at 14,000 x g for 1 minute. Empty the catch tube. Add the remaining
binding mixture to the SPIN Filter tube and centrifuge as before. Empty the
catch tube again. 
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
13. Add 500 μL of prepared Wash Buffer #2 to the SPIN Filter tube and gently resuspend
using the force of the liquid from the pipette tip to resuspend the pellet. Do
not vortex. NOTE: Ensure that ethanol has been added to the Wash Buffer #2
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
14. Centrifuge samples at 14,000 x g for 2 minute. Discard the flow-through. 
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
15. Centrifuge the sample again for 2 minutes to extract residual ethanol from the binding
matrix and dry the sample. 
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
16. Transfer the SPIN Filter bucket to a clean 1.9 mL Catch Tube. Add 60-100 μL TES. Flick
the tube or stir the matrix with a pipette tip to resuspend the pellet. Do not
vortex.
SECTION: MP Biomedicals FastDNA™ SPIN Kit for feces
17. Centrifuge samples at 14,000 x g for 2 minutes to elute
purified DNA into the clean catch tube. Discard the SPIN filter. Store at -80°C until use.