They are magnetic particles coated with carboxyl groups in the form of succinic acid that can bind DNA non-specifically and reversibly. If added to the DNA in the presence of polyethylene glycol PEG and salt usually NaCl , they replace the capricious gel extraction with standardized and quick, binding and elution steps.
How do SPRI beads work? Basic chemistry will not help you explain why negatively charged carboxyl groups have an affinity for the also negatively charged DNA. It is through the complex and not yet fully understood effects of PEG and high salt concentration on the structure and hydration of the DNA that binding is possible. The selective purification of libraries leaving in solution contaminating adaptors and adaptor-dimers comes from the ability to control the low-end of the DNA sizes that are bound to the beads.
In general, the higher the concentration of PEG and salt in the solution, the shorter the cutoff size, and therefore the lower the starting molecular weight of the purified products. Since the library fragments will always be longer than the size of the two adaptors at the ends of the DNA insert, fine-tuning the concentrations of these compounds to exclude the characteristic size of adaptor-dimers from binding to the beads will in effect purify your library.
Therefore, in practice you can calibrate the concentration of PEG and NaCl in your experiments by varying the ratio of DNA sample volume to the bead volume you add for an example of how to do this see the Keats lab web page. Make sure that the DNA sample you use for the calibration a DNA molecular weight ladder for example is in the same buffer as your library, or calibration might fail!
In an alternative library preparation protocol, sheared DNA is bound to SPRI beads and all subsequent steps are performed on-bead rather than in-solution Fisher et al. Disclaimer: Beckman Coulter makes no warranties of any kind whatsoever express or implied, with respect to this protocol, including but not limited to warranties of fitness for a particular purpose or merchantability or that the protocol is non-infringing. All warranties are expressly disclaimed. Your use of the method is solely at your own risk, without recourse to Beckman Coulter.
Not intended or validated for use in the diagnosis of disease or other conditions. This protocol is for demonstration only, and is not validated by Beckman Coulter. We appreciate your patience while we continue to improve the online experience at Beckman. Contact Us. Sign in to view contract pricing Your shopping cart is empty. Qty Price View Cart. Added to cart View Cart. Explore PCR Models. Fill in your details below or click an icon to log in:.
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