Centrifugation Of Microspin Columns For The Purification Of Nucleic Acids

With the current fast development of genomic studies

, the demand for safe, economical, high-throughput processes and equipment for nucleic acidisolation and purification has also increased.A variety of MicroSpin* column kits containing specifically designed filters or membranes are commerciallyavailable for this application.

MicroSpin kits can be used with centrifugation to obtain plasmids, PCR* (polymerase chain reaction) amplified DNA, or double-stranded replicates from microbial cultures (such as E. coli or phage liquid cultures) or mammalian sources (such as blood, tissue, and cells). These minipreps efficiently purify nucleic acids such that subsequent steps (sequencing, enzymatic restriction,RNA transcription, transfection, or microinjection) can be carried out without interference from contaminants (proteins, RNA, polysaccharides, undesirable residual PCR products, and metabolites).

MicroSpin kits are available in the form of singlecolumn or tubes with filters or membranes in the format of strips of 8 (or 12) columns or tips, as well as in the format of standard 96-well microtiter plates. The 8 or 12 strips can be supported with deep-well or square-well plates. The filter membrane containing a large-pore, silica-based matrix/gel coated witha high density of anion exchange groups is used to capture nucleic acids onto the filter assembly. These disposable assemblies are generally spun from100 x g to 30,000 x g for 15 seconds to 30 minutes, depending on the specific protocol in the separation and purification process.

A typical process involves the following steps where separation can be achieved by centrifugation.

" Pelleting""The cell is pelleted (1,500 x g for 15 minutes at 4C) and then resuspended in buffer or pretreatment reagent (i.e., protease,RNase) to obtain a cleaner prep.

" Lysis and Clarifying""The cell suspension is subjected to alkaline lysis followed by neutralization. The cell lysate is clarified to remove cell debris (up to maximum speed or 20,000 x g to " 30,000 x g for 10 to 30 minutes at 4C).

" DNA Binding""Nucleic acids are selectively adsorbed to the silica membrane (" 6,000 x g to 15,000 x g for 1 to 2 minutes).

" Washing and Extraction (in a solution containing ethanol for DNA precipitation, 6,000""15,000 x g for 30 minutes at room temperature or 4C).

" Elution (" 6,000 x g for 2 to 5 minutes). Purified (desalted and concentrated) nucleic acids are collected for further processing(i.e., restrictive enzyme analysis, amplification and sequencing steps).

Of note, when working with small (microliter) volumes, tapping the side of the tube (or whole plate) or a brief low-speed spin (i.e., " 100 x g for 2 minutes) will ensure that the droplets adhering to the side wall of the tube will be collected toward the bottom for complete mixing and better recovery. It is recommended to avoid the critical speed range for each rotor cited in its particular rotor manual during the low-speed spin. For applications requiring 500 to 1,200 rpm (the critical speed of the JS-5.3 rotor), we recommend running the rotor a few hundred rpm above or below critical speed if vibration is experienced.

Reference- https://www.beckmancoulter.com/wsrportal/bibliography?docname=A-1926AFinal.pdf

by: Scott eric

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