The increasing use of recombinant DNA methods has spawned a wide variety of plasmid purification methods. Extractions employing alkali, phenol, or boiling have been used in the past for the purification of small amounts of DNA. However, these methods are often time consuming and sometimes require dangerous solvents such as phenol and chloroform.
NucleoSpin is a user friendly purification system made especially for small amounts of plasmid DNA by Macherey-Nagel, a company that was established very early in this century and that has pioneered the manufacturing of separations media for different biomolecules.
The NucleoSpin system uses specially treated membranes that can bind up to 20 ”g of plasmid DNA. Each membrane comes in a micro spinning cup that can be placed in standard 1.5 or 2.0 ml micro centrifuge tubes.
The NucleoSpin procedure is quick and easy to perform. Following a modified Birnboim and Doly modified alkaline lysis, the cleared supernatant containing the plasmid DNA is loaded onto the membrane in the spinning cup. The chaotropic salt in buffer A3 ensures that the nucleic acid binds to the membrane. After a brief washing step in order to remove contaminants like proteins and small RNA molecules the plasmid DNA is eluted in a low salt buffer such as TE ready for a variety of manipulations including restriction analysis, sequencing, amplification, or bacterial transformation. Preps on average can be completed in 20 minutes or less.
The NucleoSpin Extract system uses the same specially treated membranes as the NucleoSpin system. However, the NucleoSpin Extract kit uses a unique set of buffers that permit either the purification of PCR products or the recovery of DNA from agarose gel slices. As with NucleoSpin, no organic solvents are required and, additionally, prior removal of mineral oil is not necessary.
The NucleoSpin Extract procedure is also quick and easy to perform. Preps on average can be completed in 20 minutes or less. The DNA fragments are eluted in a low salt buffer such as TE and are ready for a variety of manipulations including restriction analysis, ligation, sequencing, or labelling.
The NucleoSpin Blood or NucleoSpin C&T also use the same specially treated membranes as the NucleoSpin system. However, each kit uses a unique set of buffers and detergents that permit the rapid and efficient purification of genomic DNA from blood or cells and tissues, respectively. DNA is free of pigment and is extremely clean.
Addenda for NucleoTrap and NucleoSpin Applications Guide
Yield will drop off dramatically if the pH is not 8.5 or greater. For nucleic acids greater than 10 kbp, yields can be improved if the elution buffer is preheated at 60C with a pH of 8.5.
Alternatively, one could incubate the spin cups and micro centrifuge tubes (with buffer) in a water bath for 10 minutes at 55C while shaking gently every two to three minutes to distribute the warm buffer across the membrane surface.
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
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