Recovery of DNA from agarose and polyacrylamide gels (electroelution to dialysis bags)

Summary

This technique (McDonell et al., 1977) allows the recovery of double-stranded DNA from agarose or polyacrylamide gel slices over a wide range of molecular masses with high yields. This method is somewhat cumbersome in that the gel sections must be placed individually in a dialysis bag and therefore cannot be used to recover multiple DNA samples. However, electroelution works well enough to avoid the problems encountered with other methods. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.

Operation method

Recovery of DNA from agarose and polyacrylamide gels (electroelution to dialysis bags)

Principle

This technique (McDonell et al., 1977) allows the recovery of double-stranded DNA from agarose or polyacrylamide gel slices over a wide range of molecular masses with high yields. This method is somewhat cumbersome in that the gel sections must be placed individually in a dialysis bag and therefore cannot be used to recover multiple DNA samples. However, electroelution works well enough to avoid the problems encountered with other methods.

Materials and Instruments

Restriction endonuclease DNA samples
Ethanol EB or SYBR Gold stain Phenol Chloroform Sodium acetate TBE Electrophoresis buffer
Agarose or polyacrylamide gel Cooked dialysis bags Dialysis bag clips Horizontal electrophoresis tank Portable long-wave UV lamps

Move

I. Materials

1. Buffers and solutions

Ethanol

EB or SYBR Gold staining solution

Phenol: Chloroform (1:1 , V/V)

Sodium acetate (3 mol/L, pH 5.2)

0.25X TBE electrophoresis buffer

0.25X TBE electrophoresis buffer with 0.5 μg/ml ethidium bromide

2. Enzyme and buffer

restriction endonuclease

3. gels

Agarose or polyacrylamide gels

4. nucleic acids and oligonucleotides

DNA samples

5. Specialized equipment

Boiled dialysis bag

Dialysis bag clips

Horizontal Electrophoresis Cells

Portable long-wave UV lamp (302 nm)

II. Methods

1. At least 100 ng of target DNA fragments can be obtained by digestion. Separate by electrophoresis in an agarose gel or polyacrylamide of appropriate concentration and stain with ethidium bromide or SYBR Gold containing 0.5 μg/ml ethidium bromide. The location of the desired bands is determined with a hand-held long-wavelength UV lamp.

2. Using a sharp scalpel or razor blade, cut agarose or polyacrylamide gel sections containing the desired bands and place on a square paraffin film moistened with 0.25X TBE. Minimize the volume of agarose cut to minimize DNA contamination by inhibitors and to shorten the distance the DNA migrates out of the gel, as well as to ensure that it can be easily placed in the dialysis bag.

3. After cutting the strips, photograph them to obtain a record of the eluted strips.

4. wearing gloves, seal one end of the dialysis bag with a dialysis bag clip. Fill the dialysis bag with 0.25X TBE until agitated, hold the bag by the neck and gently squeeze the bag to open it. Use a medication spoon to transfer the gel slice to the buffer-filled dialysis bag.

5. Allow the gel slice to sink to the bottom of the bag. Remove most of the buffer, leaving enough liquid to keep the gel slice in contact with the buffer at all times. Then clamp the bag with the dialysis bag clamp right above the gel slice to avoid air bubbles and crushing the gel slice. Mark the name of the DNA fragment on the dialysis bag clip with a marker.

6. Immerse the dialysis bag in a horizontal electrophoresis bath containing a shallow layer of 0.25X TBE. Use a glass rod or pipette to prevent the bag from floating while orienting the gel slice parallel to the electrode. Pass an electric current through the bag, usually at 7.5 V/cm, for 45-60 min. Use a hand-held, long-wave UV lamp to monitor the migration of DNA from the gel sections.

If the electrophoresis time is too short, only a portion of the DNA will migrate out of the gel slice, which will reduce the recovery rate. Similarly, if the electrophoresis time is too long, it will cause DNA to adhere to the walls of the dialysis bag. Typically, about 85% of the 0.1-2.0 kb DNA fragments can be eluted from the gel slices by electrophoresis in 0.25X TBE buffer at a voltage of 7.5 V/cm for 45-60 min.



7. Invert the current polarity electrophoresis for 20 s to release the DNA from the wall of the tube. Disconnect the power supply and remove the dialysis bag from the electrophoresis bath, rubbing it gently to allow the DNA to enter the buffer.

8. After reverse electrophoresis, open the dialysis bag clamp and carefully transfer all the buffer around the gel into a plastic tube. Remove the gel sections from the bag and stain them as described in step 9. Rinse the dialysis bag with a small amount of 0.25X TBE using a Pasteur pipette and add the wash solution to the plastic tube.

9. Immerse the gel sections in 0.25X TBE containing ethidium bromide (0.5 μg/ml) and stain at room temperature for 30-45 min. examine the gel sections under UV light to confirm that all DNA has been eluted.

10. Purify the DNA fragments by DEAE-SepHacel, or by chromatography with commercially available resins, or by phenol-chloroform extraction and standard ethanol precipitation.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/