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DNA samples may be concentrated by extraction with isobutanol. Add slightly more than one volume of isobutanol, vortex vigorously and centrifuge to separate the phases. Discard the isobutanol (upper) phase, and extract once with water-saturated diethyl ether to remove residual isobutanol. The nucleic acid then may be ethanol precipitated as described above.

E. Notes on phenol extraction of nucleic acids

The standard and preferred way to remove proteins from nucleic acid solutions is by extraction with neutralized phenol or phenol/chloroform. Generally, samples are extracted by addition of one-half volume of neutralized (with TE buffer, pH 7.5) phenol to the sample, followed by vigorous mixing for a few seconds to form an emulsion. Following centrifugation for a few minutes, the aqueous (top) phase containing the nucleic acid is recovered and transferred to a clean tube. Residual phenol then is removed by extraction with an equal volume of water-saturated diethyl ether. Following centrifugation to separate the phases, the ether (upper) phase is discarded and the nucleic acid is ethanol precipitated as described above.

A 1:1 mixture of phenol and chloroform also is useful for the removal of protein from nucleic acid samples. Following extraction with phenol/chloroform, the sample should be extracted once with an equal volume of chloroform, and ethanol precipitated as described above.

Restriction enzyme digestions are performed by incubating double-stranded DNA molecules with an appropriate amount of restriction enzyme, in its respective buffer as recommended by the supplier, and at the optimal temperature for that specific enzyme. The optimal sodium chloride concentration in the reaction varies for different enzymes, and a set of three standard buffers containing three concentrations of sodium chloride are prepared and used when necessary. Typical digestions included a unit of enzyme per microgram of starting DNA, and one enzyme unit usually (depending on the supplier) is defined as the amount of enzyme needed to completely digest one microgram of double-stranded DNA in one hour at the appropriate temperature. These reactions usually are incubated for 1-3 hours, to insure complete digestion, at the optimal temperature for enzyme activity, typically 37degC. See the Appendix for a listing of restriction sites present in the M13 (pUC) MCS and a listing of various restriction enzymes, incubation conditions and cut sites.

Protocol

1. Prepare the reaction for restriction digestion by adding the following reagents in the order listed to a microcentrifuge tube:

*If desired, more than one enzyme can be included in the digest if both enzymes are active in the same buffer and the same incubation temperature.

Note: The volume of the reaction depends on the amount and size of the DNA being digested. Larger DNAs should be digested in larger total volumes (between 50-100 ul), as should greater amounts of DNA.

Refer to the vendor's catalog for the chart of enzyme activity in a range of salt concentrations to choose the appropriate assay buffer (10X High, 10X Medium, or 10X Low Salt Buffers, or 10X SmaI Buffer for SmaI digestions). Restriction enzymes are purchased from Bethesda Research Laboratories, New England Biolabs, or United States Biochemicals.

2. Gently mix by pipetting and incubate the reaction at the appropriate temperature (typically 37degC) for 1-3 hours.

3. Inactivate the enzyme(s) by heating at 70-100degC for 10 minutes or by phenol extraction (see the vendor's catalog to determine the degree of heat inactivation for a given enzyme). Prior to use in further protocols such as dephosphorylation or ligation, an aliquot of the digestion should be assayed by agarose gel electrophoresis versus non-digested DNA and a size marker, if necessary.

Agarose gel electrophoresis (2) is employed to check the progression of a restriction enzyme digestion, to quickly determine the yield and purity of a DNA isolation or PCR reaction, and to size fractionate DNA molecules, which then could be eluted from the gel. Prior to gel casting, dried agarose is dissolved in buffer by heating and the warm gel solution then is poured into a mold (made by wrapping clear tape around and extending above the edges of an 18 cm X 18 cm glass plate), which is fitted with a well-forming comb. The percentage of agarose in the gel varied. Although 0.7% agarose gels typically are used, in cases where the accurate size fractionation of DNA molecules smaller than 1 kb is required, a 1, 1.5, or 2% agarose gel is prepared, depending on the expected size(s) of the fragment(s). Ethidium bromide is included in the gel matrix to enable fluorescent visualization of the DNA fragments under UV light. Agarose gels are submerged in electrophoresis buffer in a horizontal electrophoresis apparatus. The DNA samples are mixed with gel tracking dye and loaded into the sample wells. Electrophoresis usually is at 150 - 200 mA for 0.5-1 hour at room temperature, depending on the desired separation. When low-melting agarose is used for preparative agarose gels, electrophoresis is at 100-120 mA for 0.5-1 hour, again depending on the desired separation, and a fan is positioned such that the heat generated is rapidly dissipated. Size markers are co-electrophoresed with DNA samples, when appropriate for fragment size determination. Two size markers are used, phi-X 174 cleaved with restriction endonuclease HaeIII to identify fragments between 0.3-2 kb and lambda phage cleaved with restriction endonuclease HindIII to identify fragments between 2-23 kb. After electrophoresis, the gel is placed on a UV light box and a picture of the fluorescent ethidium bromide-stained DNA separation pattern is taken with a Polaroid camera.

Protocol