Electrophoresis Of Restrictiondigested

DNA Nucleic acids are negatively charged at a neutral pH, which allows their migration through an electric field (8). Agarose is a highly porous polysaccharide that acts as a sieve, allowing the fragments of DNA to be separated according to length. Under low-voltage conditions, the electrical resistance of all components remains constant and the linear DNA fragments move through the agarose gel at a velocity proportional to the voltage applied. The driving force for nucleic acid migration in the gel is the voltage gradient, which is dependent on the geometry of the electrophoresis chamber, geometry and composition of the gel, and the volume and ionic strength of the buffer used. Decreasing the distance between electrodes, decreasing gel thickness, or decreasing buffer volume can increase the velocity of DNA migration. A practical approach is to keep the gel geometry, buffer composition, and volume constant and determine the optimal running voltage empirically. The gel should be covered by 3-4 mm of buffer and highvoltage settings should be avoided, because they will lead to melting of the agarose and the appearance of artifacts on the final blot. The gel can be run overnight (12-16 h) at a low

Digested Plasmid Agarose Gel

Fig. 1. Agarose gel electrophoresis of plasmid restriction digests. Five micrograms of a 3.9-kb plasmid containing a 400-bp insert of the c-met gene were digested with the restriction enzyme BstXl for 1 h at 37°C. Lane A contains DNA molecular-weight standards (ffiradlII-digested X DNA). Lane B contains plasmid that was not incubated with a restriction enzyme. Lane C contains plasmid that was incubated at a termpera-ture of 4°C instead of 37°C, demonstrating the importance of incubation temperature. Lane D contains fully digested plasmid DNA. Note the disappearance of the 3.9-kb DNA band corresponding to the intact plasmid, the appearance of a 400-bp DNA band representing the insert, and a 3.5-kb DNA band representing the plasmid remnant. The very high-molecular-weight band present in lanes B and C is likely to consist of aggregates of circular plasmid DNA and disappears with enzymatic digestion (lane D).

Fig. 1. Agarose gel electrophoresis of plasmid restriction digests. Five micrograms of a 3.9-kb plasmid containing a 400-bp insert of the c-met gene were digested with the restriction enzyme BstXl for 1 h at 37°C. Lane A contains DNA molecular-weight standards (ffiradlII-digested X DNA). Lane B contains plasmid that was not incubated with a restriction enzyme. Lane C contains plasmid that was incubated at a termpera-ture of 4°C instead of 37°C, demonstrating the importance of incubation temperature. Lane D contains fully digested plasmid DNA. Note the disappearance of the 3.9-kb DNA band corresponding to the intact plasmid, the appearance of a 400-bp DNA band representing the insert, and a 3.5-kb DNA band representing the plasmid remnant. The very high-molecular-weight band present in lanes B and C is likely to consist of aggregates of circular plasmid DNA and disappears with enzymatic digestion (lane D).

Electrophoresed Genomic Dna Bands

Fig. 2. Agarose gel electrophoresis of genomic DNA restriction digests. Genomic DNA samples were isolated from cultures of rat liver epithelial cells. Lane A contains DNA molecular-weight standards (Hindlll-digested X DNA). Lanes B and C show 10-|lg DNA samples that were digested with either Hindlll (B) or BamH1 (C) for 18 h at 37°C. Agarose gel ecectrophoresis was carried out for 18 h at 22 V. The gel was stained with ethidium bromide and photographed under an ultraviolet lamp. The distinct bands (indicated with arrows) within the digested DNA represent repetitive sequences or elements in the DNA. Note that this banding pattern differs depending on the restriction enzyme employed. Thorough digestion of the DNA produces a "smear" of DNA fragments that range in size from very large (>23 kb) to very small (<0.5 kb).

Fig. 2. Agarose gel electrophoresis of genomic DNA restriction digests. Genomic DNA samples were isolated from cultures of rat liver epithelial cells. Lane A contains DNA molecular-weight standards (Hindlll-digested X DNA). Lanes B and C show 10-|lg DNA samples that were digested with either Hindlll (B) or BamH1 (C) for 18 h at 37°C. Agarose gel ecectrophoresis was carried out for 18 h at 22 V. The gel was stained with ethidium bromide and photographed under an ultraviolet lamp. The distinct bands (indicated with arrows) within the digested DNA represent repetitive sequences or elements in the DNA. Note that this banding pattern differs depending on the restriction enzyme employed. Thorough digestion of the DNA produces a "smear" of DNA fragments that range in size from very large (>23 kb) to very small (<0.5 kb).

voltage (20-30 V) without compromising the quality of elec-trophoretic separation. Because the electrophoresed DNA should be transferred to a solid support (nylon or nitrocellulose) as soon as possible, overnight electrophoresis is often a desirable option for workers with time limitations.

Agarose electrophoresis of DNA allows separation of fragments ranging from 200 to 1 x 107 basepairs (bp), although it is not possible to separate such a wide range of lengths on a single gel. A classical Southern analysis (as presented in Table 1) allows separation of fragments ranging from 200 bp to 20 kbp. Fragments smaller than 200 bp are typically analyzed by utilizing polyacrylamide gels (9-11), and DNA larger than 20 kbp can be analyzed by pulsed-field gel elec-trophoresis (12).

The percentage and composition of agarose used to prepare the gel are determined based on the size of the frag-ment(s) of interest. Good electophoretic separation of small DNA fragments (0.2-1.0 kbp) can be accomplished using 2-4% agarose gels prepared with a 3:1 mixture of low-melting-point agarose and standard agarose (FMC BioProducts, Rockland ME). Low-melting-point agarose consists of hydroxyethylated agarose, which has better sieving properties than standard agarose and results in greater clarity of DNA bands. These high-percentage gels are useful when analyzing PCR products or cloned DNA. For Southern analysis of genomic DNA, 0.7-1.2% standard agarose gels are recommended (7). The efficiency of DNA transfer to a solid support is increased with decreasing agarose concentration, but low-percentage agarose gels are delicate and difficult to manipulate. The ideal sample well size should be determined empirically. Although a weak signal can be amplified by

Table 1 Southern Blot Analysis

1. Restriction enzyme digestion a. Digest 10-20 |lg genomic DNA with an appropriate enzyme (use 3-5 U enzyme/|lg DNA).

b. Check the efficiency of the digest by analyzing a 1-|g aliquot of DNA on an agarose gel.

c. Precipitate the remaining digested DNA overnight with 1/10 vol of 2.5 M sodium acetate and 2 vol cold ethanol (100%)

d. Resuspend precipitated DNA in 30 |L of 1X TPE and add 6 |L of of DNA sample buffer (Table 2).

2. Electrophoresis of the DNA

a. Prepare a 0.9% agarose gel with TPE buffer (add ethidium bromide to 0.5 |g/mL).

b. Place the gel in the electrophoresis tank and fill with TPE to 3-4 mm above gel surface.

c. Load the samples into the sample wells; include appropriate DNA size standards.

d. Run the gel overnight at 22-30 V (or until the bromophenol blue migrates 8 cm).

e. Photograph the gel and carefully measure migration distances of molecular-weight standards.

3. Denaturation and neutralization of the DNA

a. Denature the DNA by soaking the gel 2X 15 min in 0.5 M sodium hydroxide.

b. Neutralize the DNA by soaking the gel 3X 10 min in 1.0 M Tris-HCl pH 7.5.

4. Transfer of the DNA to a nylon membrane (alkaline method)

a. Cut a piece of nylon membrane to the exact size of the gel; prewet in dH2O

b. Assemble the capillary transfer apparatus as shown in Fig. 3. Take care to remove any air bubbles between the gel and the nylon membrane.

c. Fill buffer reservoirs with alkaline transfer buffer (Table 2).

d. Tranfer for 1-3 h (use 3 h to ensure transfer of large DNA fragments).

e. Check the efficiency of transfer by staining the gel with ethidium bromide f. Let the membrane air-dry completely; or let the membrane air-dry briefly then fix the DNA to the filter by ultraviolet crosslinking.

5. Hybridization with labeled nucleic acid probe a. Prepare the probe utilizing manufacturer's instructions b. Prehybridize the membrane for 1 h in prehybridization solution option 1 (Table 2), at 42°C.

c. Hybridize the membrane overnight in hybridization solution option 1 (Table 2), at 42°C.

d. Wash the membrane 2X 15 min in 2X SSPE, 0.1% SDS, at 42°C. If additional washing is needed, wash for 30 min in 1X SSPE, 0.1%

SDS, at 42°C. If necessary, subsequent washes can be performed (3X 10 min) with 0.5X SSPE, 0.1% SDS, at 42°C.

6. Visualization (radiolabeled probes): Rinse the membrane briefly in 1X SSPE, blot excess fluid from the membrane, wrap securely in plastic wrap, and expose to X-ray film for 24 h at -70°C. Develop the film and adjust exposure time as necessary.

decreasing the width of the sample well, the use of wider sample wells results in better resolution of bands.

The inclusion of a DNA size standard on analytical DNA gels containing DNA fragments of known length is recommended because such standards provide a means of extrapolating the size of a positive signal from target DNA. Formerly, a popular choice for DNA size standard in a classical Southern blot has been lambda phage DNA digested with the restriction enzyme HindIII, which provides a pattern of fragments ranging from 125 bp to 23.1 kb. Although, various DNA size standards are commercially available containing a greater number of DNA bands (Amersham [Arlington Heights, IL], Invitrogen) and can be obtained prelabeled with molecules, such as biotin, that aid in their visualization. When choosing DNA standards, it is important to be sure that the target DNA sequences are within the range of kilobase lengths represented in the DNA standards. Most protocols recommend staining the electrophoresed agarose gels with ethidium bromide to visualize the DNA standards and the digested DNA. Gels can be stained after electrophoresis by soaking in a 2-|g/mL solution of ethidium bromide. Alternatively, ethidium bromide (0.5 |g/mL) can be added to the melted agarose (after cooling to 55°C) and to the elec-trophoresis buffer. Staining with ethidium bromide permits photography of the gel, so the exact migration of DNA standards can be recorded along with the quality of the restriction enzyme digestion of the test DNA.

After electrophoresis, the double-stranded DNA fragments must be denatured into single strands. Denaturation of the DNA can be accomplished by soaking the gel in an alkaline solution containing sodium hydroxide (see Table 2). This step could be carried out on a rotary platform, which allows thorough, constant submersion of the gel. After denaturation, it is important to neutralize the gel, which is typically done by soaking the gel in a neutral (pH 7.4) solution of Tris buffer. The single strands of DNA are then ready to be transferred to a solid support, such as nitrocellulose or nylon membrane, where they can be hybridized to a complementary, labeled nucleic acid probe.

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