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that are highly supercoiled where conformational stresses cause
small sections of single stranded DNA to be exposed. This removes
all supercoiling and leaves a single linear form of the plasmid that
runs true to size [ 2 ]. The concentration of many different forms of
the plasmid into a single linear entity enables visualization of the
DNA using ethidium bromide staining when illuminated by UV
light. Plasmid sizes can then be estimated against a DNA molecular
ladder. The presence and position of any gene in the plasmid or
chromosome of each strain is then visualized by autoradiography
after probing with the respective radiolabeled gene.
2 Materials
All solutions are prepared using autoclaved double distilled water.
- 1/10 TE buffer; 1 mM Tris–HCl, 0.1 mM EDTA, pH 8.
Prepare 1× TE buffer: To 10 mL of Tris 1 M, pH 8, add 2 mL
of 0.5 M EDTA, pH 8 and then dilute to 1000 mL with water.
Dilute 5 mL of 1× TE to 50 mL to produce 1/10× TE
buffer. - 1× S1 buffer: 30 mM sodium acetate pH 4.6, 1 mM ZnSo4,
5% glycerol. Prepare 10× S1 buffer by adding 12.3 g of sodium
acetate and 0.92 g Zinc acetate to 200 mL of water. Add
250 mL of glycerol and adjust pH to 4.6. Then add water to
500 mL. Dilute 5 mL of 10× S1 buffer to 50 mL to produce
1× S1 buffer. Store 10× buffer aliquoted at − 20 °C. - Gel and gel running buffer 0.5× TBE: 45 mM Tris–HCl,
45 mM boric acid, 1 mM EDTA. Prepare 10× TBE buffer by
adding 108 g Tris base, 55 g boric acid and 40 mL 0.5 M
EDTA, pH 8 to 700 mL of water. Adjust pH to 8 with concen-
trated HCl and make up to 1000 mL and autoclave before use.
Add 100 mL of 10× TBE to 1900 mL of water to produce
0.5× TBE. - Denaturing solution: 0.5 M NaOH, 1.5 M NaCl. Add 20 g
NaOH and 87.66 g NaCl to 1 L water. - Neutralizing solution: 0.5 M Tris–HCl, pH 7.5, 1.5 M NaCl.
Dissolve 60.5 g Tris base and 87.6 g NaCl in 800 mL of water
adjust to pH 7.5 with concentrated HCl. - The prehybridization solution consists of (6× SSC, 0.1%
(W/V) polyvinylpyrrolidone, 0.1% Ficoll, 0.5% SDS, 150 μg/
mL herring testes DNA, and 1 mL UHT full cream milk made
up to 20 mL with water. - Random priming: Prime-It II Random Primer Labeling Kit,
Agilent Technologies, UK.
2.1 S1 Digestion
Components
2.2 Gel Running
Components
2.3 In Gel
Hybridization
Components
Mark A. Toleman