Antibiotic Resistance Protocols (Methods in Molecular Biology)

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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. 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.


2. 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.


3. 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.


4. Denaturing solution: 0.5 M NaOH, 1.5 M NaCl. Add 20 g
   NaOH and 87.66 g NaCl to 1 L water.


5. 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.


6. 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.


7. 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