Purification Of Dna Fragments From Agarose Gels

Elution of DNA Bands by High-Speed Centrifugation of Agarose Gel Slices

We have developed a rapid, simple method for eluting DNA bands from regular agarose gels by high-speed centrifugation. The agarose matrix containing DNA of interest will be compressed by the force of centrifugation, releasing DNA into the supernatant fluid. The method allows recovery of large as well as small DNA molecules. In addition to being rapid and simple, this method is of low cost and it does not require the use of phenol and chloroform as compared with traditional methods. The eluted DNA is of high quality and is suitable for restriction enzyme digestion, ligation, cloning or labeling without further purification.1 The protocol is described next:

1. Carry out electrophoresis as described in Chapter 7.

2. When electrophoresis is complete, quickly locate the DNA band of interest by illuminating the gel on a long wavelength (>300 nm) UV transilluminator. Quickly slice out the band of interest using a sharp, clean razor blade.

Note: To avoid potential damage to the DNA molecule, the UV light should be turned on as briefly as possible.

3. To enhance the yield of DNA, trim away extra agarose gel outside the band and cut the gel slice into tiny pieces with the razor blade on a clean glass plate.

4. Transfer the fine slices into a 1.5-ml microcentrifuge tube (Eppendorf).

Notes: (1) If it is not necessary to maximally elute DNA out of the gel slices, the slices do not need to be further sliced into tiny pieces. They can be directly placed in a tube. (2) At this point, there are two options for eluting DNA from the agarose gel pieces. The first is to carry out high-speed centrifugation (step 5) immediately. The second option is to elute the DNA as high yield as possible (see below).

5. Centrifuge at 12,000 to 14,000 x g or at the highest speed using an Eppendorf centrifuge 5415C (Brinkmann Instruments, Inc.) for 15 min at room temperature.

Principles: With high-speed centrifugation, the agarose matrix is compressed or even partially destroyed by the strong force of centrifugation. The DNA molecules contained in the matrix are released into the supernatant fluid.

6. Following centrifugation, carefully transfer the supernatant fluid containing DNA into a clean microcentrifuge tube. The DNA can be used directly for ligation, cloning, and labeling as well as restriction enzyme digestion without ethanol precipitation. Store the DNA solution at 4 or -20°C until use.

Tips: (1) In order to confirm that the DNA is released from the gel pieces, the tube can be briefly illuminated with long wavelength UV light after centrifugation. An orange-red fluid color indicates the presence of DNA in the fluid. (2) The supernatant fluid should be immediately transferred from the agarose pellet; within minutes, the temporarily compressed agarose pellet may swell, absorbing the supernatant fluid.

High Yield and Cleaner Elution of DNA

1. Add 100 to 300 ml of dd.H2O or TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to the gel pieces at the preceding step 4.

Function: TE buffer or dd.H2O is added to the gel pieces to help elute the DNA and simultaneously to wash EtBr from the DNA during high-speed centrifugation.

2. Centrifuge at 12,000 to 14,000 x g at room temperature for 20 min. When the centrifugation is complete, briefly visualize the eluted DNA by illumination using a long-wavelength UV light.

3. Immediately transfer the supernatant that contains the eluted DNA into a fresh tube.

4. To increase the recovery of DNA, extract the agarose pellet by resuspend-ing the pellet in 100 ml of dd.H2O or TE buffer followed by another cycle of centrifugation.

5. Pool the DNA supernatant and precipitate the DNA with ethanol. Dissolve DNA 10 ml of dd.H2O or TE buffer. Store the DNA solution at 4 or -20°C until use.

Elution of DNA Fragment by Melting and Thawing of Agarose

Gel Slices2,3

1. After restriction enzyme digestion, carry out electrophoresis as described in Southern blotting in Chapter 7, except use 0.5 to 1% low melting-temperature agarose instead of normal agarose.

2. Transfer the gel to a long-wavelength UV transilluminator (305 to 327 nm) to visualize the DNA bands in the gel. Excise the band area of interest with a clean razor blade and transfer the slices into a microcentrifuge tube.

Notes: (a) The gel should not be placed on a short-wavelength (e.g., <270 nm) UV transilluminator because this type of UV light may cause breakage inside the DNA fragment. (b) The gel slices containing the DNA band of interest should be trimmed as much as possible in order to remove excess unstained gel areas.

Caution: UV light is harmful to the human body. Protective eye glasses, gloves, and a lab coat should be worn.

3. Add two volumes of TE buffer to the gel slices and completely melt the gel in a 60 to 70°C water bath.

Note: The gel slices can be directly melted without adding TE buffer. The DNA concentration is usually higher, but the total yield of the DNA fragments is much lower than that if TE buffer is added.

4. Immediately chill the melted gel solution on dry ice or its equivalent and leave the tube at -70°C for at least 20 min.

5. Thaw the gel mixture by vigorously tapping the tube. It takes 5 to 10 min to thaw the gel into a resuspension state.

6. Centrifuge at 12,000 x g for 5 min at room temperature.

7. Carefully transfer the liquid phase containing the eluted DNA fragments into a fresh tube, which can be used directly for labeling, although the concentration is low.

8. Extract EtBr with three volumes of water-saturated n-butanol.

9. Precipitate the DNA by adding 0.1 volume of 3 M sodium acetate buffer (pH 5.2) and 2.5 volumes of chilled 100% ethanol to the DNA solution. Precipitate DNA at -70°C for 1 h and centrifuge at 12,000 x g for 5 min at room temperature.

10. Discard the supernatant and briefly rinse the DNA pellet with 1 ml of 70% ethanol. Dry the DNA under vacuum for 15 min. The DNA can be dissolved in an appropriate volume of dd.H2O or TE buffer. Store at -20°C before use.

Elution of DNA Fragment Using NA45 DEAE Membrane

1. Perform step 1 in the previous section, but use 1% normal agarose.

2. Soak a piece of NA45 DEAE membrane (Scheicher and Schuell) in 10 mM EDTA buffer (pH 7.6) for 10 to 20 min at room temperature and in 0.5 N NaOH solution for 5 min. Wash the membrane with dd.H2O three to four times and store at 4°C prior to use.

3. During the electrophoresis, monitor the migration of the DNA bands stained with EtBr in the gel using a long-wavelength UV lamp. Turn off the power, make an incision in front of the DNA band of interest and insert a piece of the prepared membrane into the incision in the gel.

4. Continue electrophoresis until the DNA fragments migrate onto the membrane by monitoring the migration of the stained band.

5. Remove the membrane strip from the gel and transfer it in a solution containing 20 mM Tris-HCl (pH 8.0), 0.15 M NaCl, 0.1 mM EDTA. Rinse the membrane briefly to remove any agarose.

6. Transfer the strip into 0.2 to 0.3 ml of elution buffer containing 20 mM Tris-HCl (pH 8.0), 1M NaCl, and 0.1 mM EDTA.

7. Incubate the strip at 55 to 68°C for 30 to 60 min with occasional agitation.

8. Rinse the strip with 0.1 ml of elution buffer and extract EtBr with three volumes of water-saturated n-butanol.

9. Precipitate and dissolve the eluted DNA as described in the preceding section.

Elution of DNA Fragments in Agarose Gel Well

1. Carry out DNA electrophoresis as described in the section on elution by melting and thawing agarose gel slices, except add the running buffer up to the upper edges of the gel instead of covering the gel.

2. During electrophoresis, check the separation of DNA bands stained by EtBr in the gel using a long-wavelength UV lamp. Stop the electrophoresis and use a razor blade or a spatula to make a well in front of a specific band. Add 10 to 30 ml of running buffer into the well.

3. Continue electrophoresis until the band migrates into the well by monitoring the band.

4. Stop electrophoresis and transfer the solution containing DNA of interest from the well to a fresh tube.

5. Extract EtBr with three volumes of water-saturated n-butanol, precipitate and dissolve the DNA as described in the section on elution by melting and thawing agarose gel slices.

0 0

Post a comment