TITLE: DNA Extraction Microbiome Kit - Fecal
AUTHORS: Stephanie  Clouser

[DESCRIPTION]
DNA Extraction of fecal samples with the MagMAX Microbiome Kit from Applied Biosystems.

[STEPS]
SECTION: Before You Begin
1. 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.                                              
     i.     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.
SECTION: Before You Begin
2. 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”)
SECTION: Lyse Sample
3. 1. Add 800 uL of Lysis Buffer to bead tubes.
2.     Prepare fecal samples as follows: 



 



3.     Vortex the bead tubes upside down for 10 seconds. 
4.     Place tubes in bead ruptor for 10 minutes at 20 Hz. 
            a.     At this point bead tubes can be stored at 4°C overnight.
SECTION: Prepare Plates
4. 
 

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. Note: These solutions are 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. 


 

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. 


 


3. Prepare Sample Plate
        a. Remove tubes from bead beater.
        b. Transfer 400uL of sample to appropriate well of a deep-well plate. 
                    i. at this point, the lysate can be stored at -20C for 3 months.
        c. Invert Bead Binding Mix, then add 520uL to each sample in the sample plate. 
        d. Proceed directly to the KingFisher. 

SECTION: KingFisher Processing
5. 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. 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.