The NucleoTrap extraction kit is recommended for the purification of DNA fragments from TAE or TBE agarose gels or aqueous solutions without using organic extraction with phenol or chloroform. Alcohol precipitation of DNA is not necessary. DNA can be eluted in a small volume (20-50”l) of TE-buffer (pH 8.5) or H2O (pH 8-8.5) and is suitable for PCR*, LCR, labelling reactions, ligations or sequencing.
DNA purified with NucleoTrap or NucleoTraPCR gives excellent results in sequencing experiments using radioactive or fluorescent detection methods. The principle is based on a specially activated matrix (not glass) that binds DNA in the presence of chaotropic salts. The chaotropic agents disrupt the water complexes surrounding the DNA molecules and as a result the DNA is reversibly adsorbed to the matrix. A subsequent washing step removes all impurities including agarose, proteins, salts, and dyes quantitatively. In contrast to the adsorption, elution occurs in a low salt media (TE buffer or H2O). Extraction of DNA can be performed using normal agarose or low melting agarose. Unlike glass powder material, the homogenous size and structure of the spherical NucleoTrap particles, which are manufactured by Macherey-Nagel, allow the purification of large DNA fragments without shearing. Also unlike glass milk products the specially activated silica matrix contains no impurities. Therefore, no later inhibition of enzymatic reactions is observed.
Purification of DNA fragments NucleoTrap NucleoTraPCR
From agarose gels +++ ---
From aqueous solutions +++ +++ (desalination)
Separation of nucleotides and/or ++ +++ reagents like biotin after labelling reactions
Separation of enzymes +++ +++
For the direct purification of Amplified DNA fragments --- +++ (PCR* products) --- not recommended, + applicable with low recoveries, ++ well applicable, +++ optimal
The binding capacity of the NucleoTrap matrix for DNA molecules is extraordinarily high. This is supported by the fact that even the smallest DNA fragments (20-50 bp) can be extracted out of agarose gels with recovery rates of approximately 50%. For the isolation of DNA fragments between 500 bp to 5 kb, a recovery rate of 80% is guaranteed. For those researchers who want to purify multiple samples in a minimal amount of time, it is noteworthy that NucleoTrap is the first product to offer two different instructions for use. The high speed protocol (50% time reduction) is designed for the extraction of 400 bp to 5 kb DNA fragments. In comparison to the longer, standard protocol, the overall yield is reduced, but by no more than 10%. DNA fragments smaller than 400 bp and larger than 5 kb should be purified by using the standard protocol.
Using NucleoTrap, the extraction of DNA molecules from aqueous media can be performed very easily. Proteins, nucleotides, dyes, and other molecules are removed very efficiently. NucleoTrap also represents a potential alternative to alcohol precipitation since it can be used to remove salt from DNA solutions quickly and efficiently.
The NucleoTraPCR extraction kit is specially designed for the direct purification of PCR products without the prior requirement for gel electrophoresis. The purification principle is the same as used by NucleoTrap. Chaotropic agents Allow the DNA to be adsorbed reversibly to the matrix. In contrast to the NucleoTrap matrix, the NucleoTraPCR matrix can only bind DNA fragments >150 bp. This means that primer and primer-dimer molecules stay in solution whereas larger DNA fragments bind to the matrix. After two washing steps the DNA is eluted in TE-buffer (10mM Tris/HCl, 1mM EDTA, pH 8.5) or another slightly basic, low salt media . DNA purified in this way shows a high purity. All impurities such as enzymes, detergents, nucleotides, and primer-dimer molecules are removed efficiently. DNA can be used for all kinds of cloning experiments, ligations, labelling reactions, or sequencing. DNA purified with NucleoTraPCR gives excellent results in sequencing experiments using radioactive or fluorescent detection methods.
Addenda for NucleoTrap and NucleoSpin Applications Guide
Yield will drop of 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 60ûC 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.
Back to the Nucleobond AX applications guide