Jun 24, 2022

Public workspaceDNA Extraction Microbiome Kit - Fecal V.2

This protocol is a draft, published without a DOI.
  • 1Pennsylvania State University
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Protocol Citation: Stephanie Clouser 2022. DNA Extraction Microbiome Kit - Fecal. protocols.io https://protocols.io/view/dna-extraction-microbiome-kit-fecal-cbynspveVersion created by Stephanie Clouser
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: June 24, 2022
Last Modified: June 24, 2022
Protocol Integer ID: 65262
Abstract
DNA Extraction of fecal samples with the MagMAX Microbiome Kit from Applied Biosystems.
Materials
a.     MagMAX™ MICROBIOME Ultra Nucleic Acid Isolation Kit 
b.     200PF Molecular Grade Ethanol
c.     Nuclease-Free Water
d.     Sterile Pipette Tips (P100, P1000)
e.     Serological Pipettor
f.      Serological Pipettes (25, 50)
g.     50 mL conical tube(s)
h.     Plate Tube rack
i.      Reagent Reservoirs 
j.      5 Deep-Well Plates 
k.     2 Elution Plate
l.      1 Tip comb 
m.   Plate Films
n.     Bead tubes
o.     Zymo 1:10 (for positive control)
p.     VWR marker
q.     Scissors
r.      Plate Map (reference “2.3_Plate-Map-Template”)
Before You Begin
Before You Begin
20m
20m
Prepare your workspace
a. Turn on Biological Safety Cabinet blower, white light, and window alarm.
b. Clean the cabinet with 70% ethanol, including the work surface, walls, and glass.
c. Lower the sash and run the UV light for 15 minutes.                                             
Safety information
Do not trust the glass to protect you from UV exposure. You can be in a different part of the lab while it is running, but do not loiter in front of the cabinet.

d. Switch the UV light back to white light; you are ready to begin.
e. Clean all materials with 70% ethanol before putting them into the cabinet.
15m
Gather Materials
You can use the small projector cart to hold materials and your notes next to the cabinet. Clean cart well with 70% ethanol before use.
a. MagMAX™ MICROBIOME Ultra Nucleic Acid Isolation Kit 
b. 200PF Molecular Grade Ethanol
c. Nuclease-Free Water
d. Sterile Pipette Tips (P100, P1000)
e. Serological Pipettor
f.  Serological Pipettes (25, 50)
g. 50 mL conical tube(s)
h. Plate Tube rack
i. Reagent Reservoirs 
j. 5 Deep-Well Plates 
k. 2 Elution Plate
l. 1 Tip comb 
m. Plate Films
n. Bead tubes
o. Zymo 1:10 (for positive control)
p. VWR marker
q. Scissors
r. Plate Map (reference “2.3_Plate-Map-Template”)
5m
Lyse Sample
Lyse Sample
1h
1h
1. Add Amount800 µL of Lysis Buffer to bead tubes.
2. Prepare fecal samples as follows: 

AB
Fresh/Frozen Fecal SamplesWeigh out 100mg, then place into prepared bead tubes.
Fecal samples in storage solutionRemove 200uL, then place into prepared bead tubes.
Fecal SwabsRemove the plastic stick, then place swab into prepared bead tubes.
 
3. Place tubes in bead ruptor for 10 minutes at 20 Hz. 
4. Remove tubes from bead ruptor and centrifuge. Centrifigation12500 rpm, 25°C, 00:02:00
a. At this point bead tubes can be stored at 4°C overnight or at -20°C for up to 3 months.
2m
Prepare Plates
Prepare Plates
1h
1h

ABCD
Plate IDPlate TypeReagentVolume Per Well
Tip CombStandardPlace a 96 deep-well tip comb on a standard plate.
Sample PlateDeep WellSample + Binding Bead Mix as described in the protocol.
Wash 1Deep WellWash Buffer1000uL
Wash 2Deep WellWash Buffer1000uL
Wash 3Deep Well80% Ethanol1000uL
Wash 4Deep Well80% Ethanol1000uL
ElutionStandardElution Buffer200uL*
*Can be reduced to 100uL if using 1 fecal swab

1. Prepare mixture of Viral/Pathogen Binding Solution + DNA/RNA Binding Beads
a. Combine 500uL/sample of Viral/Pathogen Binding Solution and 20uL/sample of DNA/RNA
Binding Beads.
Safety information
The Viral/Pathogen Binding Solution is very viscous!

i. Prepare enough for 100rxns if running a full plate.
ii. Mix solution well by inversion and store at room temperature. Do not vortex or it will be too foamy to
aliquot.


AB
In each of two 50mL conical tubes (50 rxns each):
Viral/Pathogen Binding Solution25mL
DNA/RNA Binding Beads (vortex vigorously first)1mL (or 1000uL)

2. Prepare 80% Ethanol Wash Buffer
a. Make enough 80% ethanol for 2mL per sample. Check the shelf of kit reagents for leftover 80% ethanol in the
labeled flask.


AB
In a clean flask (100rxns total):
100% molecular grade ethanol (in flammable cabinet)160mL
Invitrogen Ultra-Pure Water40mL


3. Prepare Sample Plate
a. Remove tubes from bead beater.
b. Transfer Amount400 µL of sample to appropriate well of a deep-well plate.
c. Invert Bead Binding Mix, then add Amount520 µL to each sample in the sample plate.
Safety information
The Bead Binding Mix is very viscous. Use a fresh pipette tip for every well.

d. Proceed directly to the KingFisher.

1h 30m
KingFisher Processing
KingFisher Processing
35m
35m
1. Turn on the KingFisher Flex (the on switch is on the bottom left of the machine).
2. Push the right arrow to select "user" then the down arrow to select "DNA."
3. Push the down arrow until you reach "MagMAX_Microbiome_Stool_Flex" and then press "Start."
4. Load your prepared plates onto the machine following the prompts on the screen. Press the "Start" button to
advance to the next steps.
a. BE SURE THAT THE PLATE IS ORIENTED CORRECTLY, with the A1 corner of the plate. matching the A1 labeled
on the KingFisher plate dock. Make sure the plate is sitting perfectly flat and not at a slight angle.
5. Once all plates are loaded, press "Start" to begin the protocol. Record usage in the logbook next to the machine.
6. After the program completes, you will be prompted to unload your plates the same way you loaded them. Keep the
Elution plate, seal with fresh plate film, and store at 4C for short term.
Safety information
If you are storing for longterm, transfer the DNA from the Elution plate to non-stick DNA tubes and store in -80C.

7. All other plates can be discarded in the biohazard waste bin.
8. Turn off the KingFisher and wipe with DNA away.
35m