The Dynabeads MyOne™ SILANE suspension is the key component of this kit. These uniform, monosized ferrimagnetic beads are 1 μm in diameter, and are composed of highly cross-linked polystyrene with evenly distributed magnetic material. The beads are further coated, enclosing the iron oxide inside the beads and presenting a bead surface with optimized silica-like chemistry. The increased magnetic strength of these beads ensure rapid magnetic mobility and efficient isolation of DNA. The beads also feature a low sedimentation rate and favourable reaction kinetics, making them particularly suited for automated assays.
The Dynabeads MyOne™ SILANE component of this kit is available as a separate product, and can be supplied in bulk quantities on an OEM-basis.
The Dynabeads SILANE genomic DNA kit provides an excellent tool for isolation of genomic DNA from human blood, following a simple separation protocol. A buffer is first added to the sample for lysis, followed by incubation with Dynabeads MyOne™ SILANE. The beads with bound DNA are easily pulled to the side of the test tube by using a magnet (e.g. Dynal MPC™) and unbound material is removed by aspiration. The magnetic separation also facilitates simple washing and elution of the isolated DNA. Dynabeads magnetic separation technology is easily adapted to automated liquid handling platforms. Please contact Invitrogen Dynal for further information.
Additional Material Needed
- Magnetic device (See www.lifetechnologies.com/magnets for magnet recommendations)
- Mixing/rotation device
- Test tubes and pipettes
- Proteinase K at a concentration of 20 mg/ml (e.g. Sigma Cat.No. P2308, dissolved in 10 mM Tris-HCl, pH 8)
- Isopropanol (≥ 99.5%)
- Ethanol (96-100%)
Related Dynabeads Products
The Dynabeads MyOne™ SILANE component of this kit is also available as a separate product (Cat. no. 370.02D). This product is manufactured under validated production processes, and can be supplied in bulk quantities on an OEM-basis. The Dynabeads SILANE viral NA kit (Cat. no. 370.11D) contains Dynabeads MyOne™ SILANE and specific buffers optimized for sensitive isolation of viral DNA/RNA from human serum/plasma samples. A comprehensive range of Dynabeads for specific capture of nucleic acids is available, across different beads sizes and surface functionalities. Some Dynabeads are precoated with streptavidin, allowing for capture of biotinylated molecules in a wide variety of protocols. Other Dynabeads have a specific surface chemistry for coupling of nucleic acids (e.g hybridization probes/primers) and/or other ligands. Dynabeads products are sold offthe- shelf, and also supplied in bulk quantities on an OEM-basis. The many unique benefits, superior quality and unsurpassed reproducibility of all Dynabeads are renowned in the IVD market. Based on our extensive knowledge and intellectual property (IP) we are able to work with a wide range of customers and partners to develop and validate optimized Dynabeads, buffers and protocols that meet their specific target requirements (e.g. capture of specific virus), downstream applications and regulatory requirements.
The protocol described below illustrates isolation of genomic DNA from 350 μl of human blood using 50 μl (2 mg) Dynabeads MyOne™ SILANE. The protocol can be scaled up or down to suit specific needs (sample volumes, elution volumes etc).
Preparing the buffers
- Washing Buffer 1 (genomic DNA) and Washing Buffer 2 (genomic DNA) are supplied as concentrates. To obtain working solutions, isopropanol/ethanol must be added to the two buffers as indicated on the respective labels.
- Prior to first time use, add 40 ml isopropanol to each of the two supplied bottles of Washing Buffer 1 (genomic DNA) to obtain a working solution.
- Prior to first time use, add 70 ml ethanol to each of the two supplied bottles of Washing Buffer 2 (genomic DNA) to obtain a working solution.
- Proceed with isolation of genomic DNA as described below.
Isolation of genomic DNA
- Do not add Proteinase K directly into Lysis/Binding Buffer (genomic DNA).
- All vortex steps should be performed at maximum speed.
- The vial containing Dynabeads MyOne™ SILANE should be resuspended (e.g. vortex) to a homogenous suspension prior to use. Leave the vial on a roller until use.
- For adaptation to automated format the vortex steps can be replaced by pipette action.
- Add 50 μl Proteinase K (20 mg/ml) to a 350 μl blood sample. Vortex 30 seconds (all vortex steps are at maximum speed.) Incubate 2 minutes at room temperature (RT).
- Add 350 μl Lysis/Binding Buffer (genomic DNA) to the sample and vortex 15 seconds to mix. Incubate for 5 minutes at 55°C and then vortex for 15 seconds. Incubate again for further 5 minutes at 55°C followed by a final vortex for 15 seconds.
- Add 50 μl resuspended Dynabeads MyOne™ SILANE suspension and vortex for 5 seconds to mix. Add 400 μl isopropanol (100%). Vortex 30 seconds and incubate on a roller for 3 minutes at RT followed by a final vortex for 30 seconds.
- Place the tube on the magnet and let the Dynabeads collect at the magnet for 2 minutes. Remove the supernatant by using a pipette. The Dynabeads/gDNA will form a nice pellet at the side of the tube, avoid touching this pellet with the pipette.
- Remove the magnet and add 950 μl Washing Buffer 1 (genomic DNA). Vortex 30 seconds at RT. The bead pellet should easily break into barely visible aggregates.
- Place the tube on the magnet and let the Dynabeads collect at the magnet for 1 minute. Remove the supernatant.
- Repeat steps 5 and 6.
- Add 950 μl Washing Buffer 2 (genomic DNA). Vortex 30 seconds at RT to resuspend the Dynabeads, and transfer the resuspended bead-solution to a clean tube.
- Place the tube on the magnet and let the Dynabeads collect at the magnet for 1 minute (or until the supernatant is clear.) Remove the supernatant.
- Add 950 ul Washing Buffer 2 (genomic DNA) and vortex 30 seconds.
- Place the tube on the magnet and let the Dynabeads collect at the magnet for 1 minute. Remove the supernatant. (Make sure that all the supernatant is completely removed in this last washing step. Use a small pipette tip and make sure that no droplets are left on the tube wall.) Leave the tube on the magnet and let the bead pellet dry for 5 minutes at RT.
- Remove the magnet and add 100 μl Elution Buffer (genomic DNA). Completely resuspend the Dynabeads by vortexing and/or pipetting for 2 minutes at RT.
- Place the tube on the magnet and let the Dynabeads collect at the magnet for 1 minute.
- Transfer the supernatant containing the purified genomic DNA to a clean tube.