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Automated Nucleic Acid Purification Using MagMAX Kits and Instruments

The MagMAX™ Express-96 plates, deep well plates (squared wells), and PCR plates (skirted) can be used with the instrument. Each plate type has an optimized MagMAX™ Express-96 head and tips. Of the three plates, the MagMAX™ Express-96 plate can be used with all three kinds of magnets. The volume of the deep well plates has to be a minimum of 2.2 mL (2.0 mL capped) with squared wells.

Yes. Both deep well plates and MagMAX™ Express-96 plates can be used during the same run. Therefore, it is possible to start the processing by using larger volumes (in a deep well plate) and elute the purified sample to a smaller volume (in a MagMAX™ Express96 plate).

We offer a complete MagMAX™ Express-96 system called the MagMAX™ Express-96 Magnetic Particle Processor for the purification and processing of proteins, DNA, RNA, and cells in a 96-well format. The processor handles particles automatically according to the preloaded purification protocols.

The MagMAX™ Express 96 magnets are made of material that is very stable. The magnetic field will not get weaker. However, extreme mechanical force or heating can cause damage to the magnets. Note: It is very important to keep the MagMAX™ Express-96 heads away from each other and other magnets at all times. Clashing of the magnets together can cause serious damage to the magnets.

The MagMAX™ Express-96 system should not be kept in close proximity to magnetic tapes, computer discs, or other magnetic storage systems, such as credit cards, as these can be damaged by the strong magnetic field of the MagMAX™ Express-96 heads. Do not hold the MagMAX™ Express-96 heads close to a PC display, since this may cause damage to the display. Do not use metal tools when handling MagMAX™ Express-96 heads.

Warning: This product contains very strong permanent magnets. People wearing a pacemaker or metallic prosthesis should not use this product. A pacemaker or prosthesis may be affected or damaged if it comes in close contact with a strong magnetic field.

The heating block is located inside the instrument and can be used automatically during the protocol. Both MagMAX™ Express-96 plates and PCR plates (skirted) can be heated using specially designed, interchangeable heating blocks. Any number of heating steps can be added to the protocol. During the protocol, when the protocol enters the heating step, the plate is automatically moved to the dedicated heating position for heating. After the heating step, the protocol continues automatically to the next step.

Note: No cooling steps can be added.

We strongly recommend that you keep the specified volumes within the defined limits to avoid spillover of the reagents.

Mixing with the tip comb is designed to occur below the liquid surface. This prevents aerosols and splashing from occurring when using the MagMAX™ Express instrument.

There are two rows of 12 magnetic heads each, and 2 plates for loading.

Deep well plates are recommended. Use a deep well configuration for larger sample inputs (up to 400 μL).

Yes, you can use 0.1 mM EDTA as an alternative (though beads may pellet slower).

This table shows the reagents shared among different MagMAX™ kits:

 

AM1835

AM1836

AM1939

AM1830

AM1837

AM1839

AM1840

Lysis/Binding Solution Conc.

X

X

X

X

X

X

X

RNA Binding Beads

X

X

X

X

X

X

X

Wash Soln. 1 Conc.

X

X

X

X

X

X

X

Wash Soln. 2 Conc.

X

X

X

X

X

X

X

Elution Buffer

X

X

X

X

X

X

X

Lysis/Binding Enhancer

 

X

X

X

X

X

X

Carrier RNA

X

X

X

 

 

 

X

TURBO™ DNase

 

 

 

X

X

X

 

MagMAX™ TURBO™  DNase Buffer

 

 

 

X

X

X

 

Zirconia Bead Tubes

 

 

 

 

 

 

X

TRI Reagent®

 

 

 

 

 

X

 

RNA Rebinding Conc. or Bead Resusp. Soln.

X

 

 

x

 

 

 

*Fill volumes may differ from kit to kit; however, the same composition is used.

The MagMAX™ Magnetic Particle Processor can capture small RNAs if you adjust the binding solution and Wash Solution 1 to 50% isopropranol. The Wash Solution 2 and Elution Solution remain the same. You can expect highest miRNA recovery with the modified protocol for Cat. No. AM1839.

Sample type

Input quantity

Typical yield

Mouse brain

10 mg

10–30 µg

Mouse liver

10 mg

15–60 µg

Mouse tail

0.5 cm

10–40 µg

Mouse spleen

10 mg

15–80 µg

Cultured cells

1 x 10^5 cells

15–30 µg

Buffy coat

50 µL

5–15 µg

Blood sample

50 µL

1–3 µg

Whatman® FTA or SS 903 cards

2 punches

0.2–1 µg

Buccal swabs

1 swab

0.2–1 µg

Yes, carrier RNA can be ordered separately. The Cat. No. is 4382878.

Yes, adjust the binding solution and wash solution 1 to 50% isopropanol to precipitate small RNAs. However, we have not yet tested yield with this protocol modification.

Yes, the Applied Biosystems™ MagMAX™ mirVana Total RNA Isolation Kit (Cat. No. A27828) will isolate both small and large RNAs from a variety of samples (serum, plasma, blood, tissue, cells, urine).

It is a personal preference as to which magnetic stand to purchase. Cat. No. AM10027 can be easier if using a pipettor for liquid handling, as beads clump to the side, Cat. No. AM10050 if using a robotic liquid handler, as beads clump in a ring.

Yes, it comes with one processing plate, two elution plates, and four plate covers to support manual processing. However, you will have to purchase deep-well plates, standard plates, and tip combs in addition to that if you wish to process on a KingFisher ™ instrument.

Yes, you can, but we only recommend storing the lysed samples in Lysis Binding Mix at -20 degrees C for up to 4 days. Samples should be thawed to room temperature before proceeding with the RNA purification.

Yes, xylene can be used as well. Citrisolv Clearing Agent is a healthier alternative to xylene as it does not need to be handled under a fume hood.

RNAse digestion is not needed because the DNA binding step has been optimized so that minimal RNA is pulled into the DNA fraction. For most downstream applications, some residual RNA should not cause any interference.

The MagMAX FFPE DNA/RNA Ultra Kit allows for the sequential isolation of RNA and DNA from the same FFPE sample. The MagMAX FFPE Total Nucleic AcidIaolation kit requires you to perform separate protocols for RNA and DNA extraction using separate FFPE sections.

We recommend using the MagMAX FFPE DNA/RNA Ultra Kit since it is the most versatile and optimized kit. In addition to just isolating RNA or DNA, it allows for sequential isolation of RNA and DNA from the same FFPE sample.

No, the buffers of the two kits are quite different and cannot be used interchangeably.

Yes. We recommend using sections of 10 μm or thicker for miRNA extraction.

Protease Digestion Buffer, Binding Solution, and DNA Wash Buffer are also available as standalone Cat. No. A32796. For processing sections that are thicker than 40 μm, additional reagents would be required.

Yes, we have a protocol for sequentially isolating DNA and RNA from the same FFPE sample.

We highly recommend using the elution buffer that is included in the kit. Our R&D team has tested nuclease-free water for elution and has found it not to be as efficient resulting in more bead clumping and lower RNA/DNA yield.

Yes, we offer the AutoLys M Tube system, which creates cleared lysates from FFPE tissue samples without the need for deparaffinization or wash steps, while increasing DNA and RNA yields.

The AutoLys M Tube system has only been tested with the MagMAX FFPE DNA/RNA Ultra Kit, but it may also work with our RecoverAll Total Nucleic Acid Isolation Kit for FFPE .

The FFPE-associated wax and debris are held in the upper chamber of the AutoLys M tube while the lysate passes through. Nucleic acids can then be purified from the clarified lysate. The AutoLys M tubes thus reduce manipulation of the sample eliminating the need to pellet the tissue and wash, so tissue loss is minimized

No, they have to be purchased separately together with some other products that are necessary for using the AutoLys M Tube system efficiently:
- AutoLys M Tubes and Caps (Cat. No. A38738)
- Autolys M Tube Locking Lid (Cat. No. A37954)
- AutoLys M TubeLifter (Cat. No. A37956)
- AutoLys M Tube Pliers (Cat. No. A38261)
- AutoLys M Tube Racks (Cat. No. A37955)

No, the AutoLys M Tubes can be lifted manually, but the tools significantly reduce hand strain and increase ease of use as well as throughput.

Any centrifuge with plate adapters will work. The AutoLys M Tube Racks fit nicely into those adapters.

No. The AutoLys M tubes don't fit in a standard 1.5 mL or 2.0 mL heat block. We recommend incubating the tubes in the tube racks in an incubator or oven.

No, the 96 well standard and 96 Deep Well heads are not interchangeable. However, the same tip combs can be used for both instruments.

Yes, the MagMAX™ Express-96 instrument can connect to a computer with the most recent BindIt™ software v3.3.1.7 installed.  A serial port to USB converter is required to connect.  Existing .kf2 files will not be editable in the BindIt™ software but it is possible to re-write a .kf2 file as a .bdz file so that it is editable. Please see this application note for detailed instructions on how to do that.

The MagMAX DNA Multi-Sample Ultra Kit is optimized to isolate genomic DNA (gDNA) from a variety of samples, such as whole blood, buccal cells, saliva, urine, blood cards, mouth rinse, and tissue.

Yes, you can combine the two elution buffers prior to eluting the DNA; instead of having the instrument pause, requiring you to add DNA Elution Buffer 2, press ‘start’ on the instrument - just be sure to combine them directly before adding to the elution plate and before putting the plate on the KingFisher instrument (you do not want them to sit as a mix for too long). For example, if you wish to elute in 50 µL of buffer, you can mix 25 µL of DNA Elution Buffer 1 and 25 µL of DNA Elution Buffer 2, then add the total 50 µL into a well and run the protocol as you normally would.

RNase A treatment is not critical for this workflow. RNA that might be present in the samples is likely to be degraded in the course of the experiment and should not interfere with downstream applications. For those sample types that might potentially have some RNA carryover, we have included the RNase A as part of the Bead mix as a precaution.

DNA Elution Buffer 1 is at a basic pH to help maximize release of the gDNA from the magnetic beads and has to be neutralized by DNA Elution Buffer 2.

We recommend using one of the following types of buccal swabs with foam tips:

- Puritan PurFlock Ultra Flocked Swabs (Fisher Scientific, Cat. No. 22-025-192)
- Puritan HydraFlock Swabs, standard tip (Puritan, Cat. No. 25-3306-H )
- Sterile Foam Tipped Swabs (Puritan, Cat. No. 25-1506 1PF)
- 4N6FLOQSwabs, regular tip (Thermo Fisher Scientific, Cat. No. 4473979)

Note: Please do not use cotton swabs, as they may contain PCR inhibitors.

Buccal swabs can be shipped at 25 degrees C or below. Exposure of the swabs to high temperatures (over 30 degrees C) during shipment has been associated with poor performance in the workflow. To prevent degradation, ensure that buccal swabs are completely dry before shipment. Place the swabs in a paper envelope for storage after collection - paper envelopes are permeable and allow the swabs to dry effectively.

We recommend transferring the lysate to a new MagMAX Express-96 Deep Well Plate (Cat. No. 4388476). This option eliminates contamination risks and saves time. To transfer lysates, set a multi-channel micropipetor to ˜300 µL and transfer one row at a time. Each well should contain 200-250 µL after transfer.

Heparin is not recommended as an anti-coagulant since it can cause inhibition of PCR reactions. Instead, we strongly recommend collecting blood samples in EDTA or sodium citrate.

On rare occasions, the sample may turn cloudy after adding the Multi-Sample DNA Lysis Buffer. The sample will clear up when isopropanol is added. This will not have any impact on the yield or quality of the DNA.

We have optimized protocols for extracting DNA from buccal swabs, whole blood, blood cards (Whatman FTA), oral rinse, saliva, and urine samples. In addition, the kit can be used to process vaginal microbiota samples (e.g., ThinPrep, Surepath) for gDNA extraction from yeast, bacteria (Gram positive/negative), viruses, and protozoa with the addition of additional enzymatic steps.

Yields from hemolytic plasma, lipemic plasma, or other compromised samples types vary greatly from donor to donor. We recommend processing these types of samples using the manual protocol.

Levels of cell-free DNA in circulation can range from 1 ng/mL to 100 ng/mL of plasma or serum depending on the donor. For samples containing low levels of cell-free DNA, we recommend increasing the starting sample volume.

The MagMAX Cell-Free DNA Isolation Kit has been designed to isolate cell-free DNA (cfDNA) from plasma, serum, and urine samples.

No, the MagMAX Cell-Free DNA Isolation Kit contains the Dynabeads MyOne SILANE magnetic beads. For this reason we recommend using the DynaMag magnets (Cat. Nos. 12302D, 12321D).

For NGS applications, we recommend using K2EDTA tubes. For other applications, Streck Cell-Free DNA Blood Collection Tubes can also be used.

NGS, qPCR, and ddPCR are all compatible with the MagMAX Cell-Free Total Nucleic Acid Isolation Kit. The kit is specifically designed for Ion Torrent liquid biopsy panels (Oncomine Lung Cell-Free Total Nucleic Acid Research Assay, Cat. No. A35864) that require cell-free DNA as well as cell-free RNA. The minimum yield requirement for these NGS panels is 2 ng/µL, which is why the protocol includes a rebinding step that allows for lower elution volume (15 µL) and yields a more concentrated nucleic acid.

No, Proteinase K treatment is required to lyse the exosomes, which contain most of the cell-free RNA in plasma samples; it also degrades proteins.

Yes, the MagMAX Cell-Free Total Nucleic Acid Isolation Kit does isolate miRNA.

The Qubit Fluorometer cannot discriminate between cell-free DNA and gRNA, so running an Agilent High Sensitivity DNA analysis chip is highly recommended.

cfRNA concentrations in plasma are low, and the conformation/length is not compatible with the dye in the Qubit RNA HS assay. We recommend using an m1 TaqMan assay with an appropriate amplicon length to detect cfRNA, such as Hs_99999905_m1 GAPDH (122 bases), Hs99999903_m1 ACTB 171 bases or another m1 assay target that is appropriate for your sample. The m1 designation indicates that the probe spans an exon-exon boundary ensuring that signal is only generated from a template with correctly spliced exons. The assay will not detect signal from the cfDNA that is also present in the eluate. We recommend using SuperScript VILO Master Mix (Cat. No. 11755050) for the RT step and the reaction volume for this step can be scaled down to 10 µL total, using 2 µL of purified cfNA input. We recommend performing the qPCR reaction according to the protocol for TaqMan Universal Master Mix II, no UNG (Cat. No. 4440040), with sample input up to 10% of the total reaction volume.

The MagMAX CORE Nucleic Acid Purification Kit offers pre-mixed, room-temperature reagents, which greatly improve product handling and storage. Specifically, the CORE kit decreases processing times by having pre-mixed washes that do not require the addition of alcohol, a single protocol adapted on multiple KingFisher magnetic particle processors, and a complete protocol that addresses 14 unique sample types.

You can find the .bdz files located within the Documents section of the MagMAX CORE product page under "Product Literature".

The MagMAX CORE Mechanical Lysis Module is optimized for isolating nucleic acid from difficult to-lyse targets such as mycobacterium (specifically Mycobacterium avium paratuberculosis and tuberculosis), spores, and complex samples used for microbiome testing.

The size of the bead tubes that come with these two kits is different, but the bead beating settings are the same. The Mechanical Lysis Module contains a workflow for up to 5 g of feces in addition to the standard 0.3 g protocol.

The only difference is that the MagMAX CORE Mechanical Lysis Module & Glass Microbeads Bundle with CORE Chemistry (Cat. No. A37488) contains glass microbeads, whereas the other kit (Cat. No. A37487) does not. The beads are also available as a standalone item (Cat. No. A37489) and aid in the homogenization of completely desiccated fecal or environmental samples. These glass microbeads are added to the desiccated sample together with some deionized water, vortexed, and incubated for about 10 minutes prior to the bead beating step.

Table 1 of the manual lists all the different plants we have tested with the kit and the approximate DNA yields to expect.

If higher DNA concentration is needed, the elution volume can be reduced to 50 µL when using the KingFisher Flex instrument. However, we have not tested this and the script may have to be changed accordingly.

PVP adsorbs polyphenols, removing them from the final eluate. DTT, a reducing agent, protects the DNA from degradation by preventing the oxidation of polyphenols. We generally recommend adding PVP for woody, lignified, or polyphenol samples (branches, twigs, needles, and wax-coated leaves). DTT can be helpful when working with seeds. Some seeds (e.g., seeds with a pesticide coat) do benefit from addition of PVP to Lysis Buffer A. If DTT supplementation to Lysis Buffer A was already attempted and has not helped, try again with PVP instead.

Automated Nucleic Acid Purification using KingFisher Kits and Instruments

There are 4 magnetic head options available for the KingFisher™ Flex Magnetic Particle Processor:

- KingFisher™ Flex head for MagMAX™ Express-96 Deep Well plate

- KingFisher™ Flex head for KingFisher™ 24 Deep Well plate<br/>

- KingFisher™ Flex head for KingFisher™ 96 plate<br/>

- KingFisher™ Flex head for PCR plate, skirted

The consumables for the KingFisher™ Flex instrument with the 96 Deep Well head are the same as for the MagMAX™ Express-96 instrument. They are listed below: 

- MagMAX™ Express-96 Deep Well Plates (Cat. No. 4388476)

- MagMAX™ Express-96 Standard Plates (Cat. No. 4388475)

- MagMAX™ Express-96 Deep Well Tip Combs (Cat. No. 4388487)

- MagMAX™ Express-96 Standard Tip Combs (Cat. No. 4388488)

- MicroAmp™ Clear Adhesive Film (optional): (Cat. No. 4306311)

For the KingFisher™ Flex instrument with the 24 Deep Well head, the following consumables are required:

- 24 Deep-Well Plates for KingFisher™ Flex: (Cat. No. 95040480)

- 24 Deep Well Tip Combs for KingFisher™ Flex: (Cat. No. 97002610)

No, we strongly recommend that you use the MagMAX™ plates or KingFisher™ plates since they were specifically designed to be used with KingFisher™ Flex tip combs to attain maximal performance. Plates from other manufacturers may not be compatible with the KingFisher™ Flex heating blocks. They may also cause unexpected problems, such as cross-contamination, due to the divergent well volume and bottom height of the plate.

Yes, reaction volumes can be scaled up, and all other volumes (i.e. wash volumes) will have to be increased by the same fold difference. It is generally not recommended to scale up reaction volumes more than 2-fold unless the plate's volume capacity will allow it.

Yes, this can be done by sample splitting. Set the protocol so that the magnet will collect samples from multiple wells before continuing to the next step.  Be sure not to overload.

For nucleic acid extraction, we recommend using our MagMAX™ kits and MagJET kits. Other magnetic beads should work well as long as they are 0.5 to 10 µm in size. However, you will have to write your own KingFisher™ protocols using the Thermo Scientific™ BindIt™ software and validate/optimize the protocol on your own.

BindIt™ software is compatible with Windows™ XP (Professional with Service Pack 3), Windows™ Vista (Service Pack 2), Windows™ 7 (Service Pack 1), and Windows™ 8.

No, manufactured protocols are locked for editing, but they can be renamed using the “save as” function and edited as a new version.

Protocols cannot be transferred back from the KingFisher™ Flex instrument to the computer.  Protocols need to be previously stored in the BindIt™ software to make edits.

KingFisher™ Flex uses a USB port, whereas the MagMAX™ Express- 96 requires a RS-232 port.

The KingFisher™ Flex magnets are made of material that is very stable. The magnetic field will not get weaker. However, extreme mechanical force or heating can cause damage to the magnets.

Caution: It is very important to keep the KingFisher™ Flex heads away from each other and from other magnets at all times. Clashing together of the magnets may cause serious damage to the magnets.

The KingFisher™ Flex magnets are made of material that is very stable. The magnetic field will not get weaker. However, extreme mechanical force or heating can cause damage to the magnets.

Caution: It is very important to keep the KingFisher™ Flex heads away from each other and from other magnets at all times. Clashing together of the magnets may cause serious damage to the magnets.

This can be done by running the Change magnet protocol under the Maintenance menu using the “up” and “down” cursor keys.

Both deep-well plates and KingFisher™ 96 plates can be used during the same run. Therefore, it is possible to start the processing by using larger volumes (in a deep-well plate) and elute the purified sample to a smaller volume (in a KingFisher™ 96 or MagMAX™ 96 plate).

The heating block warms up at the rate of about 10 degrees C per minute, so it will take about 6 minutes.

The transport locks are only necessary when relocating the instrument.

Set up the protocol on the BindIt™ software. After the first 8 plates are done, the instrument will pause and instruct you to switch out the plates.

No, the processing volume refers to the maximum volume that can be held in the 24 Deep Well plates. This incorporates the volume of binding buffers and beads, so when scaling up reaction volumes, it is important to make sure that the total volume does not exceed 5 mL.

Yes, it comes with both the KingFisher Flex 96 KF Heating Block and KingFisher Flex 96 Deep-Well Heating Block.

Yes, here are the catalog numbers for standalone heating blocks:

  • Cat. No. 24075420: KingFisher Flex 96 KF Heating Block
  •  Cat. No. 24075430: KingFisher Flex 96 Deep-Well Heating Block
  • Cat. No. 24075440: KingFisher Flex 24 Deep-Well Heating Block

Yes, both the KingFisher Flex 96 KF and 96 Deep-Well heating blocks can be used for the 96 Deep-Well Head, but they are only compatible with certain MagMAX/KingFisher plates.

- The KingFisher Flex 96 KF Heating Block is compatible with both standard plates and Deep-Well plates (Cat. Nos. 97002540, 4388475, 95040460, 4388476).
- The KingFisher Flex Deep-Well Heating Block is only compatible with Deep-Well plates (Cat. Nos. 95040460, 4388476).

We strongly recommend that you maintain the specified volumes within the defined limits to avoid spillover and to help ensure the best performance at the most efficient level.

Automated Nucleic Acid Purification Using the iPrep System

Yes, the iPrep PureLink Total RNA Kit will do this. When prompted on screen, you could select the "No DNase" option. This will result in collection of all nucleic acids present in the sample. However, this kit was not optimized to purify DNA, so there would be preferential binding for RNA over DNA due to the GITC in the lysis/binding buffer.

The iPrep Purification Instrument allows for automated purification of nucleic acids from up to 13 samples (12 samples and 1 positive control). You will need to purchase an iPrep Kit, which contains the sample preparation reagents and reagents, as well as the iPrep Protocol Card, which is preprogrammed with the purification protocol that directs the volume of reagents used and incubation time.

iPrep Kit

Recommended iPrep™ Card

iPrep ChargeSwitch gDNA Tissue Kit

iPrep gDNA Blood and Tissue Card

iPrep ChargeSwitch Forensic Kit

iPrep Forensic Card (includes buccal protocol)

iPrep ChargeSwitch Buccal Cell Kit

iPrep Forensic Card (includes buccal protocol)

The iPrep Purification Instrument has been discontinued but we still offer iPrep purification kits and pre-programmed iPrep protocol cards to go along with the instrument.