John M. S. Bartlett.pdf - Bio-Nica.info

Detection of Nucleic Acids 69
2.2.5. Nonradioisotopic Detection and Quantitation
The increasing trend in modern molecular applications is away from the use of large
amounts of radioactivity and to substitute biotinylated or digoxigenin-labeled DNA
for 32-P- or 33-P-labeled nucleotides. More recently, directly fluorescently labeled
nucleotides have been released which, with the appropriate systems, can also aid
detection of nucleic acid. These approaches are largely applicable in northern and
southern blotting procedures, and although these can be applied to sequencing (blotting
of sequences) or other DNA gel procedures, they are time consuming and introduce an
additional potential source of error. Other methods, such as silver staining, are more
attractive because they do not require blotting, but they are generally less sensitive than
radioisotopic procedures. With this proviso, they provide a useful and rapid means of
detecting nucleic acids in situ, before proceeding with experiments.
2.3. Summary
It is of course impossible to detail all possible systems for detection of DNA in a
simple chapter such as this one; however, we have included below examples of the
major approaches to detection of nucleic acids that can be adapted to suit most needs.
This is very much a general guide for broad application and other methods can be
sourced from the references included below.
3. Materials
3.1. Agarose Gel Electrophoresis
1. Low melting point agarose (Sigma).
2. 50× TAE: 242 g of Trizma Base (Sigma) and 100 mL of 0.5 M EDTA (pH 8.0) are
dissolved in 800 mL distilled water and autoclaved. Then, 57.1 mL of glacial acetic acid
is added; make up to 1 L.
3. Gel loading buffer: 0.25% Bromophenol blue (w/v), 0.25% xylene cyanol FF (w/v), and
30% glycerol (v/v) in distilled water (see Note 1).
4. Molecular weight markers (e.g., 100-bp ladder; Gibco).
5. Gel apparatus and power pack.
6. Ethidium bromide (10 mg/mL) in water (see Note 2).
7. Ultraviolet light box and camera.
8. Activated charcoal.
3.1.1 Recovery of DNA from Agarose Gels
1. Scalpel blades.
2. β-agarose and buffer (Calbiochem, UK).
3. Phenolchloroform 11 mixture (Sigma, UK).
4. Ammonium acetate (7.5 M).
5. 100% Ethanol.
3.1.2. PAGE
3.1.2.1. NONDENATURING PAGE
1. 40% Acrylamide (38 g of acrylamide, 2 g of bis-acrylamide, Bio–Rad, UK) in distilled
water.