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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
Product Performance
The major advantages of using Dynabeads® DNA DIRECT Universal for isolation of PCR-ready DNA are listed below.
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.
Please read this section before starting your DNA isolation protocol
Critical Steps
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.
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.
Problem | Cause | Possible Solution |
---|---|---|
A complex does not form or the complex is small and fragmented. | This indicates that very little DNA is present or the DNA is degraded. |
|
The complex is unusually large and is difficult to handle.. | This indicates that there is a large amount of DNA present. |
|
The complex is difficult to break up to give a homogeneous suspension. | Either the complex has been insufficiently pipetted, or the aperture on the pipette tip is too large. |
|
PCR amplification is not observed. | This may indicate the presence of PCR inhibitors or that there are insufficient PCR cycles to achieve a product. | If the former possibility is suspected:
If the latter possibility is suspected: Increase the number of PCR cycles. |
The number of PCR reactions possible is lower than expected. | If fragmentation of the complex was observed during the washing steps, washing may have been too vigorous. If fragmentation was not observed, elution may have been inefficient. | If the former possibility is suspected:
If the latter possibility is suspected:
|
The PCR background is too high. | The concentration of the template-DNA, PCR-primers or Mg2+/ dNTPs may be too high or the isolated DNA may still contain contaminants. | If the former possibility is suspected:
If the latter possibility is suspected:
|
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.
For Research Use Only. Not for use in diagnostic procedures.