- Maxwell® HT 96 gDNA Blood Isolation System (Cat.# A2670, A2671)
- 47.5–50% ethanol (Note: Prepare by diluting 95–100% USP/ACS- or molecular biology-grade ethanol with an equal volume of molecular biology-grade water. Using denatured ethanol that contains methanol or isopropanol may decrease DNA purity or yield.)
- Pipettes and tips for filling plates with extraction reagent (Note: Use of repeater pipettes can increase efficiency when filling plates.)
System requirements: KingFisher™ Flex Purification System equipped with the KingFisher™ Flex Head for Microtiter Deep Well 96 plate and Heating Block for Microtiter 96 Deep Well Plate. The BindIt™ Software Package provided with the KingFisher™ Flex will also be required to transfer the Maxwell® HT 96 gDNA Blood v1.0 method to the instrument. To download the Maxwell® HT 96 gDNA Blood v1.0 method, contact Technical Services: firstname.lastname@example.org.
The following consumables are required for each automated extraction:
- KingFisher™ 96 Tip Comb for Deep Well Magnets (ThermoFisher Scientific Cat.# 97002534)
- KingFisher™ Deep Well 96 Plate, V-bottom, polypropylene (ThermoFisher Scientific Cat.# 95040450) – 6 plates per extraction
Caution: We recommend the use of gloves, lab coats and eye protection when working with these and any chemical reagents.
Sample handling recommendations: For fresh blood, we recommend storing at 4°C for no longer than 48 hours prior to processing. For frozen storage of blood, we recommend storing at –80°C for no longer than one month prior to processing for extraction of gDNA.
Import the Maxwell HT gDNA Blood v1.0 method into the BindIt™ Software and transfer the Maxwell HT gDNA Blood v1.0 method onto the KingFisher™ Flex as described in the Thermo Scientific BindIt™ Software Guide. The Maxwell HT gDNA Blood v1.0 method will guide you through the preparation of reagent plates for the run. The steps to prepare for a gDNA extraction from blood are outlined below.
- Ensure that the KingFisher™ Flex is set up with the KingFisher™ Flex Head for Microtiter Deep Well 96 Plate and Heating Block for Microtiter 96 Deep Well Plate. Refer to the directions in the KingFisher™ Flex User Manual to change either the Flex Head or Heating Block, if necessary.
- Load the Maxwell HT gDNA Blood v1.0 method from the Protocol Manager in the BindIt™ Software. The Protocol Manager is accessed by clicking the Open icon in the Protocol menu of the Home tab (as outlined in Figure 1, Panel A). In the Protocol Manager window that appears, select the Maxwell HT gDNA Blood v1.0 method and select “Open” to launch the method.
- Label six 96 Deep Well Plates accordingly.
Plate 1 – Elution
Plate 2 – Ethanol Wash
Plate 3 – Wash 2
Plate 4 – Wash 1
Plate 5 – Tip Plate
Plate 6 – Lysis and Bind
- Prepare Plate 5 – Tip Plate. Slide a KingFisher 96 Deep Well Tip Comb into a KingFisher Deep Well 96 Plate.
- Prepare the Plate 4 – Wash 1, Plate 3 – Wash 2, Plate 2 – Ethanol Wash and Plate 1 – Elution processing plates. Table 1 can be used to calculate the volumes of reagents to be added to each well of the processing plates. The reagent volumes to be added to the processing plates can also be found in the BindIt™ Software, as described in Figure 1, Panel B.
Note: To expedite the setup of the sample plates, we recommend that you prepare a bulk reagent mixture for each well, depending on the number of samples that are being run. A repeater pipette may be used to add the bulk mixed reagent to each well of the plate.
- Add 250µl of the blood samples to Plate 6 – Lysis and Bind Plate.
- Add the indicated volume of Lysis Buffer (CLD) and Proteinase K to the sample wells in Plate 1 – Lysis and Bind Plate. The Lysis Buffer (CLD) and Proteinase K can be added separately. Alternatively, you can prepare a bulk reagent mixture of Lysis Buffer (CLD) and Proteinase K to be added to the plate. The Prepared Binding Buffer and resin will be added to the Lysis and Binding Plate later.
If using, prepare the bulk reagent mixture of Lysis Buffer (CLD) and Proteinase K just prior (<10 minutes) to adding the mixture to your blood samples.
After the Lysis Buffer (CLD) and Proteinase K are dispensed into the wells containing blood samples, the processing plates should be loaded onto the KingFisher™ Flex and therun should be started immediately.
- Start the Maxwell HT gDNA Blood v1.0 method. If the KingFisher™ Flex is being run through a connected PC with the BindIt™ software, you can simply load the Maxwell HT gDNA Blood v1.0 method and press the Start button to begin loading the processing plates. If the instrument is being run from the internal software, find the method in the list of User Defined Protocols and press the Start button on the instrument to begin loading the plates. Place the indicated plate in the Loading Station of the instrument, then press the Start button to load the next plates until all plates are loaded. Once all plates are loaded, you can close the sliding door of the Loading Station.
- Add the Binding Buffer (BBA), RNase A and resin to each sample in Plate 1 – Lysis and Bind when prompted by the software. Prepare a bulk reagent mixture of Binding Buffer (BBA), RNase A and resin, according to your calculations in Table 1. After about 25 minutes of runtime, the software will pause and prompt for the addition of the Binding Buffer (BBA), RNase A and resin to the plate. Open the sliding door and remove the plate from the Loading Position of the KingFisher™ Flex. Add 375µl of the Binding Buffer (BBA), RNase A and resin mixture to the wells of the Lysis and Binding Plate containing sample. Return the plate to the instrument and press “Run” to resume processing.
Note: The resin must be mixed to a homogeneous suspension before addition to the bulk reagent mixture with Binding Buffer A (BBA) and resin. Also, before dispensing the Binding Buffer (BBA), RNase A and resin bulk reagent mixture to the Lysis and Bind Plate, vortex the solution for 5 seconds to ensure resin suspension. Continue to shake/vortex the mixture from time to time while adding it to the samples in Plate 1 – Lysis and Bind.
- Once the run has ended, remove the elution plate from the deck and clean up the other processing plates. Waste should be discarded per your institution’s biohazardous waste instructions. The eluate can be used immediately or stored as appropriate for later analysis.
Figure 1. Navigation in the BindIt™ Software. Panel A. Loading the Maxwell HT gDNA Blood v1.0 method. From the Home Tab, click the Protocol Open icon to open the Protocol Manager window. Select the Maxwell HT gDNA Blood v1.0 method and select Open in the Protocol Manager window to launch the method. Panel B. Determining the processing plate layout for the Maxwell HT gDNA Blood v1.0 method. Under the Layout tab, you can view each processing plate and select different plate options from the left window to see which reagents are added to each processing plate.
Table 1. Volumes of reagent to prepare for each processing plate. Thoroughly vortex all solutions of bulk reagent mixture before adding to the plate.