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New Protocols for Isolating High- Molecular-Weight Genomic DNA


Rapidly recover intact DNA from blood, bone marrow, and cultured cells

Brenda J. Rogers Mark J. Dycaico
Stratagene

Stratagene now offers updated protocols to isolate and purify high-molecular-weight genomic DNA using the RecoverEase DNA isolation kit. New procedures include DNA isolation from blood, bone marrow, and tissue culture cells. In addition, protocols for liver, lung, kidney, spleen, testes, and brain are also included. Each procedure eliminates the need for toxic phenol or chloroform and requires very little hands-on time to perform. Use the kit to minimize physical manipulation of the DNA the point in purification methodology where DNA is most susceptible to shearing forces. Fully hydrated high-molecular-weight DNA is ready to use in molecular biological applications in less than 24 hours.

The RecoverEase DNA isolation kit is designed to isolate very high-molecular-weight genomic DNA by reducing the shearing forces that take place in traditional methods using phenol-chloroform extraction and ethanol precipitation. The basic kit procedure requires tissue homogenization, filtration through a filter unit supplied with the kit to remove cellular debris (<3 minutes), isolation of the cell nuclei by centrifugation (<20 minutes), and protease digestion of the nuclei (45 minutes).1,2 The resulting suspension is transferred to a free-floating RecoverEase kit cup where the digested proteins and low molecular weight contaminants are removed by dialysis (16 to 48 hours), leaving behind fully hydrated DNA, which is ready for immediate use. From previous experiments using field inversion analysis, DNA isolated in this way, produced products that were 500-kb long.2 Subsequent Southern blot analysis demonstrated that the DNA cut with a variety of restriction enzymes (data not shown). These benefits, along w
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