Safety Procedures for Working with DNA

Safety Procedures for working with DNA

 Storage:

Ø  The following properties of reagents and conditions are important considerations in processing and storing DNA and RNA.

Ø  Heavy metals promote phosphodiester breakage. EDTA is an excellent heavy metal chelator. Free radicals are formed from chemical breakdown and radiation and they cause phosphodiester breakage. UV light at 260 nm causes a variety of lesions, including thymine dimers and crosslinks. Biological activity is rapidly lost.

Ø  320-nm irradiation can also cause crosslinks. Ethidium bromide causes photo-oxidation of DNA with visible light and molecular oxygen.

Ø  Oxidation products can cause phosphodiester breakage. If no heavy metals are present, ethanol does not damage DNA. 5°C is one of the best temperatures for storing DNA. –20°C causes extensive single- and double-strand breaks. –70°C is probably excellent for long-term storage.

Ø  For long-term storage of DNA, it is best to store it in high salt (>1 M) in the presence of high EDTA (>10 mM) at pH 8.0. Storage of DNA in buoyant CsCl with ethidium bromide in the dark at 5°C is excellent.

Purification:

Ø  To remove protein from nucleic acid solutions:

Ø  Treat with proteolytic enzyme, e.g., proteases, proteinase K.

Ø  Phenol Extract. The simplest method for purifying DNA is to extract with phenol or phenol:chloroform and then chloroform. Phenol denatures proteins and the final extraction with chloroform removes traces of phenol.

Ø  Use CsCl/ethidium bromide density gradient centrifugation method.

Quantitation:

(i) Spectrophotometric:

Ø  For a pure solution of DNA, the simplest method of quantitation is reading the absorbance at 260 nm where an OD of 1 in a 1 cm path length = 50 mg/mL for double-stranded DNA, 40 mg/mL for single-stranded DNA and RNA and 20–33 mg/mL for oligonucleotides.

Ø  An absorbance ratio of 260 nm and 280 nm gives an estimate of the purity of the solution. Pure DNA and RNA solutions have OD260/OD280 values of 1.8 and 2.0, respectively.

Ø  This method is not useful for small quantities of DNA or RNA (<1 mg/mL).

(ii) Ethidium bromide fluorescence:

Ø  The amount of DNA in a solution is proportional to the fluorescence emitted by ethidium bromide in that solution.

Ø  Dilutions of an unknown DNA in the presence of 2 mg/mL ethidium bromide are compared to dilutions of a known amount of a standard DNA solution spotted on an agarose gel or Saran Wrap or electrophoresed in an agarose gel.

Concentration:

Ø  Precipitation with ethanol DNA and RNA solutions are concentrated with ethanol as follows: the volume of DNA is measured and the monovalent cation concentration is adjusted.

Ø  The final concentration should be 2–2.5 M for ammonium acetate, 0.3 M for sodium acetate, 0.2 M for sodium chloride, and 0.8 M for lithium chloride.

Ø  The ion used often depends on the volume of DNA and the subsequent manipulations; for example, sodium acetate inhibits Klenow, ammonium ions inhibit T4 polynucleotide kinase, and chloride ions inhibit RNA-dependent DNA polymerases.

Ø  The addition of MgCl2 to a final concentration of 10 mM assists in the precipitation of small DNA fragments and oligonucleotides.

Ø  Following addition of the monovalent cations, 2–2.5 volumes of ethanol are added, mixed well, and stored on ice or at –20°C for 20 minutes to 1 hour.

Ø  The DNA is recovered by centrifugation in a microtube for 10 minutes (room temperature is okay).

Ø  The supernatant is carefully decanted making certain that the DNA pellet, if visible, is not discarded (often the pellet is not visible until it is dry).

Ø  To remove salts, the pellet is washed with 0.5–1.0 mL of 70% ethanol, spun again, the supernatant is decanted, and the pellet dried.

Ø  Ammonium acetate is very soluble in ethanol and is effectively removed by a 70% wash. Sodium acetate and sodium chloride are less effectively removed.

Ø  For fast drying, the pellet can be spun briefly in a Speedvac, although the method is not recommended for many DNA preparations, because DNA that has been overdried is difficult to resuspend and also tends to denature small fragments of DNA.

Ø  Isopropanol is also used to precipitate DNA but it tends to coprecipitate salts and is harder to evaporate since it is less volatile.

Ø  However, less isopropanol is required than ethanol to precipitate DNA, and it is sometimes used when volumes must be kept to a minimum, e.g., in large-scale plasmid preps.

Restriction Enzymes:

Ø  Restriction and DNA modifying enzymes are stored at –20°C in a non–frost-free freezer, typically in 50% glycerol.

Ø  The enzymes are stored in an insulated cooler, which will keep the enzymes at –20°C for some period of time.