Jul 07, 2022

Public workspaceExtraction of bacterial DNA using MagMAX™ CORE Nucleic Acid Purification Kit on KingFisher™ Flex Instrument V.2

DOI
dx.doi.org/10.17504/protocols.io.81wgb781ovpk/v2
  • 1University of Illinois Veterinary Diagnostic Laboratory;
  • 2Ohio Department of Agriculture Animal Disease Diagnostic Laboratory;
  • 3Mississippi Veterinary Research and Diagnostic Lab;
  • 4Oklahoma Animal Disease Diagnostic Laboratory
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DOI: dx.doi.org/10.17504/protocols.io.81wgb781ovpk/v2
Protocol Citation: Leyi Wang, Carol Maddox, Melanie Prarat, Yan Zhang, Lifang Yan, Akhilesh Ramachandran, Sai Sankara Narayanan, Girish Patil 2022. Extraction of bacterial DNA using MagMAX™ CORE Nucleic Acid Purification Kit on KingFisher™ Flex Instrument. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb781ovpk/v2Version created by Sarah Nemser
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: July 07, 2022
Last Modified: March 03, 2023
Protocol Integer ID: 66208
Keywords: Robot, KingFisher Flex, MagMAX CORE, DNA Extraction
Funders Acknowledgement:
Vet-LIRN
Grant ID: 1U18FD06983-01
Disclaimer
Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
Abstract
This procedure is used to extract genome DNA of bacteria isolates using theMagMAX™ CORE Nucleic Acid Purification Kit on the KingFisher Flex Robot. The process consists of Sample processing, Prepare plates for Robot, and Operate on KingFisher Flex machine.
Guidelines
In the whole procedure, need wear PPE
Materials
Bacterial culture isolates
MagMAX™ CORE Nucleic Acid Purification Kit (A32702 or A32700)
Safety warnings
Need work in the biosafety level 2 hood to scoop the bacteria in the buffer tube
Prepare plates for Robot
Prepare Wash Plate 2 by adding 500 μL of MagMAX™ CORE Wash Solution 2
Prepare Wash Plate 1 by adding 500 μL of MagMAX™ CORE Wash Solution 1
Prepare Elution Plate by adding 100 μL of MagMAX™ CORE Elution Buffer
Set Tip Comb in a 0.5 ml 96-Well Plate

ABCD
Plate setup of Processing Plates: KingFisher™ Flex instrument
Plate ID Plate Type Reagent Volume Per Well
Wash Plate 1 Deep Well MagMAX™ CORE Wash Solution 1 500 μL
Wash Plate 2 Deep Well MagMAX™ CORE Wash Solution 2 500 μL
Elution Plate Standard MagMAX™ CORE Elution Buffer 100 μL
Tip Comb Plate Standard Place a tip comb in the plate Place a tip comb in the plate
Prepare Bead/PK mix by adding 20 μL of MagMAX™ CORE Magnetic Beads and 10 μL of MagMAX™ CORE Proteinase K for the required number of samples plus 10% overage.
Prepare Lysis/Binding solution by adding 350 μL of MagMAX™ CORE Lysis Solution and 350 μL of MagMAX™ CORE Binding Solution for for the required number of samples plus 10% overage. Mix by inverting the tube or bottle at least 10 times.
Invert the tube of Bead/PK Mix several times to resuspend the beads, then add 30 μL of the Bead/PK Mix to the required wells in the 2.4 mL 96- deep well sample plate.
Transfer 200 μL sample to a well with 30 μL Bead/PK mix in Sample plate. Shake vigorously for 2 minutes on a plate shaker at room temperature, or pipette mix them and incubate for 2 mins at room temperature.
Add 700 μL of Lysis/Binding Solution to each sample-containing well.
Sample processing
From plate, scoop a full loop of 1-μl loop to 200 μL of PBS or molecular grade water solution. Mix the sample by vortexing.
Operate on KingFisher Flex machine
Select the right script on the instrument or load the script if it was not already in the instrument.

Download kit script KingFisher Flex heated script: MagMAX CORE_Flex.bdz at https://www.thermofisher.com/order/catalog/product/A32700#/A32700, and then use BindIt sofware to transfer this script from a computer to the machine with the connection line.
BindIt software can be requested and downloaded at the website https://www.thermofisher.com/us/en/home/global/forms/life-science/download-bindit-software-kingfisher-instruments.html
Start the run, then load the prepared plates in the appropriate positions when prompted by the instrument. The instrument will direct the user as to which plate to load in what position.
Load the Tip Comb Plate
Load the Elution Plate
Load the Wash Plate 1
Load Wash Plate 2
Lastly, load the sample plate
Click Start Button, and the machine will process and complete in around 20 mins
Laboratory specific variations from protocol

Ohio ADDL: use molecular grade water to resuspend the colonies.

Illinois VDL, Mississippi VRDL, and Oklahoma ADDL: use PBS to resuspend the colonies.

Oklahoma ADDL: uses Dry Garnet 0.7mm beads (component of QiAMP fecal kit) to lyse the colony solution on Qiagen Tissuelyzer II (Cat. No. / ID: 85300) for 90seconds at 30Hz.