3-scroll immersi in G (1,4) by Alzati A.

By Alzati A.

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Example text

Nucleic acids can be concentrated by using alcohol to precipitate the DNA or RNA from solution; the precipitate is recovered by centrifugation and can then be processed as required. 50 ␮g ml−1 for double-stranded DNA, or 40 ␮g ml−1 for singlestranded DNA or RNA. If the A280 is also determined, the A260 /A280 ratio indicates if there are contaminants present, such as residual phenol or protein. 0 for pure RNA preparations. In addition to spectrophotometric methods, the concentration of DNA may be estimated by monitoring the fluorescence of bound ethidium bromide.

If one of the dNTPs supplied is radioactive, the result is a highly labelled DNA molecule (Fig. 3). Fig. 3 Labelling DNA by nick translation. (a) A single-strand nick is introduced into the phosphodiester backbone of a DNA fragment using DNase I. (b) DNA polymerase I then synthesises a copy of the template strand, degrading the non-template strand with its 5 →3 exonuclease activity. If [α-32 P]dNTP is supplied this will be incorporated into the newly synthesised strand (solid circles). 13:16 P1: SBT 09780521850063c03 CUUS128/Nicholl 978 0 521 85006 3 March 11, 2008 WORKING WITH NUCLEIC ACIDS Fig.

These days most molecular biology supply companies sell kits that enable purification of nucleic acids from a range of sources. The first step in any isolation protocol is disruption of the starting material, which may be viral, bacterial, plant, or animal. The method used to open cells should be as gentle as possible, preferably utilising enzymatic degradation of cell wall material (if present) and detergent lysis of cell membranes. If more vigorous methods of cell disruption are required (as is the case with some types of plant cell material), there is the danger of shearing large DNA molecules, and this can hamper the production of representative recombinant molecules during subsequent processing.

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