Dynabeads DNA DIRECT Universal

Dynabeads® are uniform, superparamagnetic, monodisperse polymer particles. The uniformity of both particle size and shape allows for the rapid and efficient binding of the target to the beads. Dynabeads^® DNA DIRECT™ Universal is a complete system for the simple and rapid isolation of PCR-ready genomic DNA from small quantities of a variety of crude sample materials, e.g. clinical specimens, cultured cells and tissues from various species. A minimum quantity of mammalian sample will typically yield sufficient template for 10 PCR reactions. For the direct isolation of PCR-ready DNA from whole blood, Dynabeads® DNA DIRECT™ Blood (Prod. No. 631.02) is the product of choice.

The process of DNA isolation relies upon cell lysis and subsequent adsorption of the released DNA to the surface of the Dynabeads® during a brief incubation. This is followed by magnetic separation of the intact DNA/Dynabeads® complex, removal of the supernatant and subsequent washing to remove any residual contaminants and potential PCR inhibitors. Finally, the complex is simply resuspended for direct use in downstream PCR reactions. This cost-effective and efficient procedure is completed in a single tube in only 10 minutes. The use of Dynabeads® biomagnetic separation technology overcomes the need for time-consuming centrifugation steps and also avoids the use of hazardous chemicals that may require costly disposal. Dynabeads® DNA DIRECT

Universal is equally suited to laboratories handling small, precious samples and those with high-throughput requirements. Please refer to the reverse side of this handbook for details on automated DNA isolation using Dynabeads® DNA DIRECT Universal. Dynabeads® DNA DIRECT Universal is supplied complete with ready-to-use Dynabeads® (supplied in lysis Buffer), Washing Buffer and Resuspension Buffer.

Yield of genomic DNA isolated from blood using Dynabeads® DNA DIRECT™ Universal will depend on the number of nucleated cells present in the sample. Typically, 200 μl of Dynabeads® will isolate between 600 ng–1 μg DNA from 30 μl blood, an amount that is sufficient for 30-50 downstream PCR amplifications. The protocol can be modified for the automated isolation of PCR-ready DNA from buccal scrapes and other sample types (see section below). The DNA isolation procedure involves cell lysis, release of DNA and adsorption of DNA onto the Dynabeads® ’ surface. The intact DNA/Dynabeads® complex is then separated from the other components in solution by magnetic separation. A series of washing steps is needed to remove any residual contaminants and potential PCR inhibitors from the isolated DNA. Finally the complex is resuspended in a low ionic strength buffer for downstream PCR reactions. Dynabeads® DNA DIRECT Universal is supplied complete with ready-to-use Dynabeads® (supplied in lysis buffer), Washing Buffer, NaOH and Resuspension Buffer.

Dynabeads® DNA DIRECT Universal for automated use is based on the unique Dynabeads® biomagnetic separation technology and designed for the simple and rapid isolation of PCR-ready genomic DNA directly from blood. PCR is a rapid technique which lends itself to automation, but the isolation of genomic DNA is usually the rate limiting step. The Dynabeads® magnetic separation technology also lends itself readily to automation. Dynabeads® DNA DIRECT Universal includes all the required reagents. Dynabeads® are uniform, superparamagnetic, polymer beads. The process of DNA isolation relies upon cell lysis and the subsequent adsorption of the released DNA to the surface of the magnetic Dynabeads® in a single step. The DNA/Dynabeads® complex is pulled to the side wall of the well by applying a magnetic field (Dynal® MPC or similar). The supernatant is carefully removed. Washing to remove any residual contaminants and potential PCR inhibitors from the isolated DNA is performed in the same way. Finally, the complex is resuspended for direct use in downstream PCR reactions. Alternatively, the DNA may be eluted from the Dynabeads® with a short incubation at 65°C. The yield of genomic DNA isolated using Dynabeads® DNA DIRECT Universal will depend on the number of nucleated cells present in the sample. One unit (200 μl) of Dynabeads® applied to 30 μl of blood will isolate between 600 ng - 1 μg of high quality genomic DNA. This amount of DNA is sufficient for 30-50 PCR amplifications.

Kit Components

Dynabeads® DNA DIRECT Universal is supplied with the necessary buffers and solutions, supporting DNA-isolation from 300 samples.

  1.    Dynabeads® DNA DIRECT Universal Dynabeads® supplied in a lysis buffer. 60 ml supplied in the kit.

  2.    Washing Buffer, 10 x concentrated 100 mM Tris-HCl, pH 7.5 1.5 M LiCl 1 mM EDTA 30 ml supplied in the kit.

  3.    Resuspension Buffer 10 mM Tris-HCl, pH 8.0 30 ml supplied in the kit.

  4.   10 M NaOH 40 % (w/v) 5 ml supplied in the kit.

Warning:   The suspension is corrosive. NaOH cause burns of eyes and skin. May cause coughing, difficulty with breathing, lung damage, diarrhea or shock if inhaled or digested. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.

All components are produced and quality controlled for optimal performance and tested to be free of contaminating DNA. Material Safety Data Sheet (MSDS) is available upon request.

Storage and Stability

Provided the kit is stored correctly, all components of Dynabeads® DNA DIRECT Universal are guaranteed until the expiry date stated on the label. All components of the kit should be stored at 2-8°C. Freezing of the kit is not recommended. Precautions should be taken to ensure that DNA or microbial contamination of the kit components does not occur. Ensure proper disposal of contaminated materials and decontamination of work surfaces. The buffers and components provided with Dynabeads® DNA DIRECT Universal should be brought to room temperature and the Dynabeads® fully resuspended prior to use.

Note:   Vials containing Dynabeads® should be stored upright to ensure that the beads are covered with buffer. Drying of Dynabeads® may reduce their efficiency. If Dynabeads® do become dried, they should be resuspended by keeping the vial in motion on a roller for up to 12 hours. This should restore the functionality of the Dynabeads® .

Note:   During storage, the Dynabeads® will settle to the bottom of the bottle leaving a colorless, clear
aqueous suspension. Prior to use, the Dynabeads® should be resuspended well by gently shaking the bottle to obtain a homogeneous dispersion of Dynabeads® in solution. This will appear as an opaque light-brown suspension. Avoid foaming. Do not vortex.

Additional Materials Needed

• Liquid-handling robot or eight channel (50-200 μl) pipette with appropriate tips.
• Dynal® Magnetic Particle Concentrator (the Te-MagS from Tecan or Dynal® MPC-96 S (Cat. no. 120.27) is recommended).
• 96-well skirted PCR-plates.
• Water bath or heating block (for optional elution).

Product Performance

The major advantages of using Dynabeads® DNA DIRECT Universal for isolation of PCR-ready DNA are listed below.

• Protocols are simple, reliable and rapid.
• PCR results show excellent reproducibility in combination with a high level of sensitivity.
• Isolated DNA is of high integrity and high molecular weight (1).
• The sensitivity and efficiency of DNA capture enables successful isolation, even when the quantities of starting material are limited.

• Using Dynabeads® DNA DIRECT Universal, the requirement for centrifugation and hazardous chemicals is eliminated.

• The method enables samples to be batched, so that a large number of samples can be processed simultaneously.

• Automation of the protocol is possible.

• Using Dynabeads® DNA DIRECT Universal and the automated protocol on a Tecan Genesis® RSP (Tecan AG, Switzerland), 48 samples can be processed in 60 minutes.

Dynabeads® DNA DIRECT Universal can be used with anti-coagulated blood (using anti-coagulants such as ACD, citrate or EDTA), blood stored at room temperature or at 2-8°C for up to one week, frozen blood and buffy coat. It is possible to use sample volumes of up to 30 μl human blood.

Dynabeads® DNA DIRECT Universal has been successfully tested on blood samples from individuals with various white blood cell (WBC) counts and also on blood from other mammalian sources. Blood from other mammalian sources may vary in white blood cell content and sample size should be adjusted not to exceed 1 μg DNA per sample. Buccal scrape samples have also shown good results.

The yield of genomic DNA isolated using Dynabeads® DNA DIRECT Universal will depend on the number of nucleated cells present in the sample as well as the species the sample was obtained from. One unit (200 μl) Dynabeads® applied to 30 μl of human blood will isolate between 600 ng - 1 μg high quality genomic DNA to be used as template DNA for 30-50 PCR amplifications. For PCR amplifications, see below.

Note:   If determination of DNA concentration by absorbence measurement is required (2), DNA must first be eluted off the Dynabeads® . Ensure that there are no Dynabeads® left in the solution as the beads will interfere with the spectrophotometrical readings. The DNA concentration can also be checked by agarose gel electrophoresis. If NaOH is used, the isolated DNA will be denatured (partially single stranded) and highly available for PCR-amplification. Please be aware that single stranded DNA has a much lower binding efficiency for ethidium bromide compared to double stranded DNA.

Manual 96-well DNA Isolation with Dynabeads® DNA DIRECT Universal using Dynal® MPC-96 S and an Eight Channel 50-200 μl Pipette.

Read all sections below before starting your DNA isolation protocol.

Note:   The buffers provided with Dynabeads® DNA DIRECT™ Universal should be brought to room temperature prior to use.

Note:   Elute the supplied 10 x Washing Buffer to 1 x concentration using sterile and PCR-grade water and equipment before proceeding with DNA isolations.

Note:   During storage Dynabeads® will settle in the bottle leaving a colorless, clear aqueous solution. Resuspend Dynabeads® before use by gentle shaking to obtain a homogeneous dispersion of Dynabeads® in solution. Avoid foaming. Do not vortex.

Warning:   The detergent in the lysis buffer may precipitate when stored at 4°C. If this should happen, simply mix and slightly heat the bottle to resuspend, obtaining a homogeneous dispersion of beads in solution. Avoid foaming. Do not vortex. Please note that chaotropes like GTC may also cause precipitation of the detergent in the lysis buffer.

1. Place your blood sample into individual wells of a 96-well plate. Volumes up to 30 μl human blood can be used. For maximum DNA yield, use up to 30 μl human blood. When more than 10 μl is used, mix the lysis solution (containing the Dynabeads® ) with 1/20 volume 10 M NaOH prior to use. When adding NaOH, the Dynabeads® solution will become cloudy.
   
   Warning:   The NaOH suspension is corrosive. Wear suitable gloves and eye/face protection.


2. Transfer 170 μl Dynabeads® resuspended in lysis buffer to sample wells. The Dynabeads® /lysis solution should be added with enough force to ensure mixing. Do not mix further.
   
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3. Leave at room temperature for 5 minutes. Continuous agitation is not required and should be avoided.


4. Place the 96-well plate on the Dynal® MPC-96 S.


5. Pause for 1-2 minutes (the longer the pause, the more compact is the DNA/Dynabeads® complex, but 1 minute will be sufficient).


6. Aspirate lysate without touching the DNA/Dynabeads® complex. To achieve this it might be necessary to aspirate off center, along the wall opposite the complex. The DNA/Dynabeads® complex will have a dark brown gelatinous appearance. During this step and the washing steps, take care not to break up the complex.


7. Remove the 96-well plate from Dynal® MPC-96 S.


8. Add 200 μl Washing Buffer (1 x concentration) to the wells. The Washing Buffer must be dispensed along the wall above the complex to ensure that the complex is flushed off the wall.


9. Place the 96-well plate on Dynal® MPC-96 S.


10. Leave for 1-2 minutes or until the supernatant has cleared.


11. Aspirate Washing Buffer without touching the DNA/Dynabeads® complex. To achieve this it might be necessary to aspirate off center, along the wall opposite the complex.


12. Repeat steps 7-11 once (< 20 μl blood samples) or twice (> 20 μl blood samples).


13. Remove the 96-well plate from the Dynal® MPC-96 S.


14. Add 100 μl water, Resuspension Buffer or low ionic strength buffer (TE or similar) to the wells. The water must be dispensed along the wall above the complex to ensure that the complex is flushed off the wall.


15. Resuspend the complex by pipetting up and down (a minimum of 20 times or until suspension is fully resuspended). Resuspension is more efficient if aspiration is performed at the center of the well and dispensing is performed off center than if aspiration and dispensing are performed at the same location. This is the first step where shear forces of any degree are applied.


16. If required, the DNA can be eluted off the Dynabeads® in Resuspension Buffer, water or low ionic strength buffer by incubation at 65°C for 5 minutes. The complex must be fully resuspended before elution: The elution buffer can be pre-heated to 65°C. After incubation, place tube in magnet stand for 30 seconds and quickly pipette off supernatant and transfer to clean tube.
    
    Note:   Do not use more than 10% of DNA/Dynabeads® suspension (from step 15) as starting material for PCR (50 μl reaction volume). The presence of Dynabeads® will not adversely affect the PCR reaction. Do not use more than 50% of eluted (from step 16) DNA should be used for one PCR. If storage of DNA is required at -20°C or for more than one week at 2-8°C, elution of DNA (step 16) is recommended. If determination of DNA concentration by absorbency measurement at A260 (2) is required, DNA must first be eluted from Dynabeads® .
    
    Sample Pre-treatment
    
    Blood samples (4, 5, 6, 20): 5-30 μl (up to 1 μg DNA) fresh capillary or anti-coagulated blood (EDTA/ACD/ citrate) can be used directly in the protocol described below. Please remember that fresh blood will coagulate within minutes if an anticoagulant is not added. Heparin blood is not recommended. If used, the sample size should be limited to 5 μl with 200 μl Dynabeads® . Stored or thawed blood should be briefly mixed on a vortex mixer prior to aliquots being taken. Hypercellular samples contain high numbers of nucleated cells and are consequently richer in DNA. This excess DNA may make the DNA/Dynabeads® complex difficult to handle and the quantity of sample introduced into the procedure should be reduced. With small blood samples (< 20 μl), the number of washing steps may be reduced from three to two.
    
    Note:   The volumes mentioned here are for human blood samples. Dynabeads® DNA DIRECT has been successfully used for isolation of DNA from blood from other species (3, 10).
    
    Buccal scrapes (6): Buccal scrapes can be collected using a simple plastic scraper. Place the scraper in a microcentrifuge tube with 50 μl PBS and spin down the sample. Remove the PBS and the scraper and follow the isolation protocol described in the next three sections. There is however a volume constraint of 40 μl for total sample size. With buccal scrape samples, the number of washing steps may be reduced from three to two.
    
    Note:   Dynabeads® DNA DIRECT Universal is not suited for use with buccal swab samples
    
    Other sample types: Dynabeads® DNA DIRECT Universal has been successfully used for manual DNA isolation from a wide range of other sample materials including cultured cells (3), bone marrow (3, 7, 16, 17), mouth wash (7), buccal scrapes and sputum (6), urine (7), bile (7), feces (7, 8), bacteria (1, 9-14), algae (10), fungi (10, 15) and various tissues from plants (10) and animals (3, 10, 18, 19), including mouse tails (6).
    
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Please read this section before starting your DNA isolation protocol

Critical Steps

1. Bring all components of the Dynabeads® DNA DIRECT Universal kit to room temperature before use.


2. Ensure that the Dynabeads® and any precipitated detergent have been fully resuspended. Before use shake gently or repeatedly invert to obtain a homogeneous dispersion of Dynabeads® in solution. Do not vortex and avoid foaming. The Dynabeads should appear as a pale brown suspension.


3. Dilute the supplied 10 x Washing Buffer to 1x concentration using sterile and PCR-grade water and equipment before starting DNA isolations.


4. The DNA/Dynabeads® complex must be handled carefully. Do not break up the complex. During lysis and washing steps, shear forces caused by pipetting, vortexing or shaking must be avoided.


5. Removal of lysate should be performed carefully to avoid loss of the DNA/Dynabeads® complex, and completely to remove all contaminants.


6. The DNA/Dynabeads® complex should be washed from the well wall in one piece following addition of the Washing Buffer. Non-complexed Dynabeads® may be observed due to excess binding capacity.


7. Ensure that all Washing Buffer is carefully and completely removed after each wash.


8. Resuspension of the final washed DNA/Dynabeads® complex should be continued until the suspension appears homogeneous.


9. If elution is performed, the final solution should be clear, colorless and free of Dynabeads® .

Handling the DNA/Dynabeads® Complex

A DNA/Dynabeads® complex will form after the Dynabeads® have been added to the sample in a single, rapid pipetting action. The complex will have a gelatinous appearance. To avoid loss of material, it is important that the complex is kept intact until the Resuspension Buffer (or low ionic strength buffer of choice) is added. Do not vortex or mix further as this may damage the complex and result in a reduced yield.

Avoid drawing the DNA/Dynabeads® complex into the pipette tip when removing supernatant. Removing the supernatant in small aliquots reduces the likelihood of accidentally drawing the complex into a pipette tip. Although it is important that the DNA/Dynabeads® complex remains intact, the washing steps must be thorough enough to ensure removal of contaminants.

Washing Buffer should be added to the tube in a single, rapid pipetting action. This will swirl the complex around in the buffer without requiring any further stirring or mixing action.

After the washes, the DNA/Dynabeads® complex is broken up in Resuspension Buffer, water or low ionic strength buffer. For complete resuspension repeatedly pipette the complex up and down until the suspension appears homogeneous. Minimizing pipetting at this step will give a higher molecular weight, but high molecular weight DNA will take a very long time to dissolve properly.

Do not stir the suspension with the pipette tip, as DNA may become stuck to the tip. Avoid pipetting air into the suspension. Resuspension is easier to perform if one leaves the complex in Resuspension Buffer, water or low ionic strength buffer for a while prior to resuspension (it may be left at 2-8°C over night). Using a pre-heated buffer can also make resuspension easier. The number of pipetting steps will depend on the liquid handling robot and the sample volume used, and should be optimized. Using a Biomek and 20-30 μl blood, we recommend the complex to be pipetted 80-100 times.

DNA Isolation using Dynabeads® DNA DIRECT

Universal and Different Magnets

DNA isolation with Dynabeads® DNA DIRECT Universal can be automated by using an integrated magnet station (e.g. Te-MagS™) or performed manually using the Dynal® MPC-96 S.

1. Using the Te-MagS™ from Tecan Further information and protocols for automated DNA isolation using Dynabeads® DNA DIRECT Universal on the Tecan Genesis^® separation system can be found in the application manual from Tecan (Doc. No. 391 352).


2. Using the Dynal® MPC-96 S The manual protocol can be performed using a magnetic particle concentrator (Dynal® MPC-96 S). For optimal performance we recommend to use 96-well skirted PCR-plates. Bar magnets will collect the bead pellets at the side of the wells. Optimal working volumes of the Dynal® MPC-96 S are 5 - 200 μl.

PCR Amplifications

DNA to be used as a template for PCR amplification must be free of PCR-inhibiting contaminants. Dynabeads® DNA DIRECT Universal is designed for the direct isolation of PCR-ready genomic DNA with a purity to meet this requirement. It is recommended not to use more than 10% of the resuspended DNA/Dynabeads® complex in Resuspension Buffer (alternatively water or low ionic strength buffer of choice), or up to 50% of the eluted DNA as starting material for PCR (50 μl reaction volume). When 200 μl Dynabeads® is applied to 30 μl of human blood, between 600 ng -1 μg of high quality genomic DNA will be isolated and can be used as template DNA for at least 30 PCR amplifications. If NaOH is used, the DNA is isolated in a way making it highly available for PCR-amplification. The PCR reaction-mixtures should then be placed on the thermal cycler only after the temperature has reached 72°C, or perform hot-start PCR. If storage of DNA is required at -20°C or for more than one week at 2-8°C, elution of DNA is recommended. Some PCR reactions are very sensitive to the amount of DNA template used. In such instances, titration of the DNA/Dynabeads® complex is recommended with comparisons made between eluted and non-eluted DNA. PCR capacity is dependent upon parameters such as genome size, the complexity of the starting material etc. Generally, 200 ng mammalian DNA will provide sufficient template for at least 10 PCR reactions. It is recommended that PCR-profiles with at least 30-35 cycles are used.

Invitrogen Dynal® AS complies with the Quality System Standards ISO 9001:2000 and ISO 13485:2003.

Storage and Stability

All components are guaranteed stable until the expiry date stated on the label when stored unopened at 2-8°C. Do not freeze the kit. Store all the buffers and components at 2-8°C. Store the vial containing Dynabeads in lysis buffer (vial 2) upright to keep the beads in solution, as drying of the Dynabeads  may result in reduced performance. Dynabeads® should be fully resuspended prior to use. The Red Cell Lysis and Washing buffer are supplied as concentrates and should be diluted to their correct concentration using sterile and PCR grade water and equipment prior to use. Take care to avoid DNA and microbial contamination. Dispose contaminated materials and decontaminate work surfaces correctly.

Warnings and Limitations

Dynabeads^® DNA DIRECT™ Blood is for research use only. Dynal® magnets should not be kept in close contact with magnetic tapes, computer discs or other magnetic storage systems, as these can be damaged by the strong magnetic field. Vial 2 contains NaOH. This suspension is corrosive and appropriate protection of skin and eyes is required. Certificates of Analysis/Compliance are available upon request. Material Safety Data Sheets (MSDS) are available on our website.

DNA isolation with Dynabeads® DNA DIRECT Universal can be automated on liquid handling robots. Dynabeads DNA DIRECT Universal can also be used for manual DNA isolation of PCR-ready genomic DNA from small samples of various sample types (such as cultured cells, bacteria, feces, mouse tails, plants etc).

Dynabeads^® DNA DIRECT™ Blood (Cat. no. 631.02) is intended for the isolation of PCR-ready DNA from whole blood. When using Dynabeads® DNA DIRECT Blood, higher yields coupled with increased sensitivity and reproducibility for genomic DNA isolation and PCR amplification have been observed compared to the standard DTAB/CTAB protocol, especially for samples with low WBC counts and low concentrations of DNA. The yield of genomic DNA isolated using Dynabeads® DNA DIRECT Blood will depend upon the number of nucleated cells present in the blood sample. Typically, one unit will isolate 1-5 μg DNA from 100 μl blood, which typically is sufficient template for 100 downstream PCR-amplifications. The protocol can be modified for the isolation of PCR-ready DNA also from 500 μl whole blood.

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