I have a dsDNA biotinylated on streptavidin Dynabeads. How can I dissociate the non-biotinylated DNA strand from the biotinylated one?
There are two methods to dissociate the non-biotinylated DNA from the biotinylated DNA strand. The following protocols are based on using 20 µL of Dynabeads Streptavidin, but are scalable. Both methods may release very small amounts of complementary biotinylated strand from streptavidin. If it is critical that no biotinylated strand is released, either adopt a different biotin modification using dual biotin (two biotin groups in sequence) or covalently bind DNA to e.g., Dynabeads M-270 Carboxylic Acid.
Using heat:
- Wash the DNA coated Dynabeads in 50 µL 1 x SSC.*
- Resuspend the beads in another 50 µL of 1 x SSC Incubate at 95 degrees C for 5 mins.
- Quickly put the tube in magnet stand for 1-2 mins and transfer the supernatant to a new tube.
- The supernatant contains your non-biotinylated DNA strand.
Using NaOH:
- Wash the DNA coated Dynabeads in 50 µL 1 x SSC.*
- Resuspend the beads in 20 µl of freshly prepared 0.15 M NaOH.
- Incubate at room temperature for 10 mins. Put the tube in magnet stand for 1-2 mins and transfer the supernatant to a new tube.
- The supernatant contains your non-biotinylated DNA strand. Neutralize the probe by adding 2.2 µL 10 x TE, pH 7.5 and 1.3 µL 1.25 M acetic acid.
Wash the Dynabeads coated with biotinylated strand once with 50 µL 0.1M NaOH, once with 50 µL of B&W buffer and once with 50 µL TE buffer.
*1 x SSC (0.15 M NaCl, 0.015 M sodium citrate. Dissolve the reagents in 800 mL water. Adjust pH to 7.0 with NaOH. Adjust the volume to 1 liter with water).
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
How do I measure the binding of biotinylated molecules on streptavidin Dynabeads?
Assay the supernatant for unbound molecules. This will determine the amount of molecule bound to the Dynabeads. For nucleic acids, the concentration can be checked by OD readings, or by running a gel. For proteins, the concentration in the supernatant can be determined by a spectrometer using a protein assay like BCA. Alternatively, you can label the molecule with radioactivity or fluorescence and measure the concentration of molecule directly on the beads (former) or in the supernatant (latter).
Find additional tips, troubleshooting help, and resources within our Dynabeads Nucleic Acid Purification Support Center.
How many biotin binding-sites are there per streptavidin molecule?
Streptavidin is a protein composed of four identical subunits, each containing a high affinity binding site for biotin (K-D = 10 -15 M) . Streptavidin has the same biotin binding properties as avidin, but it has a low isoelectric point (pI=5) and no carbohydrate groups, resulting in low non-specific binding.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
What are the differences between Pierce Streptavidin Magnetic Beads and MagnaBind Streptavidin Beads?
Pierce Streptavidin Magnetic Beads are homogeneous, non-aggregating, pre-blocked, iron oxide, superparamagnetic microparticles encapsulated with a proprietary polymer surface (no exposed iron, bead size of approximately 1 (0.5-1.5) µm, concentration 10 mg/mL). Pierce Streptavidin Magnetic Beads can be used in manual or automated applications (using KingFisher 96 and KingFisher Flex instruments, for example).
MagnaBind Streptavidin Beads are also superparamagnetic particles, but are heterogeneous aggregates made of silanized iron oxide (bead size approximately 1-4 µm, concentration 5 mg/mL). MagnaBind Streptavidin Beads can only be used in manual applications.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Can I centrifuge the MagnaBind Streptavidin Beads and Thermo Scientific Streptavidin Magnetic Beads?
No, centrifuging causes the formation of irreversible aggregates which greatly reduces binding capacity.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.