May 21, 2024
MP Biomedicals FastDNA™ SPIN Kit
This protocol is a draft, published without a DOI.
- 1Moredun Research Institute
Protocol Citation: Karen Keegan 2024. MP Biomedicals FastDNA™ SPIN Kit. protocols.io https://protocols.io/view/mp-biomedicals-fastdna-spin-kit-dd3j28kn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 21, 2024
Last Modified: May 21, 2024
Protocol Integer ID: 100171
Abstract
MP Biomedicals FastDNA‱
SPIN Kit for feces
Materials
FASTDNA Spinkit for feces
MP Biomedicals FastDNA™ SPIN Kit for feces
MP Biomedicals FastDNA™ SPIN Kit for feces
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.
Centrifuge samples at 14,000 x g for 5 minutes and decant supernatant.
Add 978 μL Sodium Phosphate Buffer and 122 μL MT Buffer.
Shake vigorously or vortex briefly to mix.
Homogenize samples in the FastPrep 24 instrument at setting
6.0m/s for 40 seconds.
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.
Transfer the supernatant to a clean 2.0 mL centrifuge tube.
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.
While samples are centrifuging, add 1 mL of Binding Matrix Solution to a clean 15 mL
conical tube (not supplied).
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.
Centrifuge samples at 14,000 x g for 2 minutes. Decant the
supernatant
Wash the binding mixture pellet by gently resuspending with
1 mL Wash Buffer #1.
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.
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
Centrifuge samples at 14,000 x g for 2 minute. Discard the flow-through.
Centrifuge the sample again for 2 minutes to extract residual ethanol from the binding
matrix and dry the sample.
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.
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.