Extraction of bacterial DNA using MagMAX™ CORE Nucleic Acid Purification Kit on KingFisher™ Flex Instrument
Leyi Wang, Carol Maddox, Melanie Prarat, Yan Zhang, Lifang Yan, Akhilesh Ramachandran, Sai Sankara Narayanan, Girish Patil, Sarah Nemser, Olgica Ceric, Mothomang Oyinloye
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.
Steps
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
| A | B | C | D |
|---|---|---|---|
| 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.
