TITLE: Extraction of bacterial DNA using MagMAX™ CORE Nucleic Acid Purification Kit on KingFisher™ Flex Instrument
AUTHORS: Leyi  Wang, Carol Maddox, Melanie  Prarat, Yan  Zhang, Lifang  Yan, Akhilesh Ramachandran, Sai Sankara Narayanan, Girish Patil, Sarah  Nemser, Mothomang  Oyinloye, Olgica  Ceric

[DESCRIPTION]
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

[STEPS]
3.8. Click Start Button, and the machine will process and complete in around 20 mins
4. 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. 
2. 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.
1. Prepare plates for Robot
1.2. Prepare Wash Plate 1 by adding 500 μL of MagMAX™ CORE Wash Solution 1
1.1. Prepare Wash Plate 2 by adding 500 μL of MagMAX™ CORE Wash Solution 2
1.3. Prepare Elution Plate by adding 100 μL of MagMAX™ CORE Elution Buffer
1.4. Set Tip Comb in a 0.5 ml 96-Well Plate

 
       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      
 
1.5. 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.
1.6. 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.
1.7. 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.
1.8. 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.
1.9. Add 700 μL of Lysis/Binding Solution to each sample-containing well.
3. Operate on KingFisher Flex machine
3.1. 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
3.2. 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.
3.3. Load the Tip Comb Plate
3.4. Load the Elution Plate
3.5. Load the Wash Plate 1
3.6. Load Wash Plate 2
3.7. Lastly, load the sample plate