Recovery of DNA from agarose gels (DEAE-cellulose membrane electrophoresis)

Summary

Recovery of DNA from agarose gels can be applied to (1) mammalian cell transformation and (2) radiolabeling.

Operation method

Recovery of DNA from agarose gels (DEAE-cellulose membrane electrophoresis)

Principle

Recovery of DNA from agarose gels is accomplished by electrophoresis onto a positively charged DEAE-cellulose membrane. In this method, DNA fragments are first separated by agarose gel electrophoresis at the appropriate concentration, and then a slit is cut immediately in front of the target DNA fragment band. A long strip of DEAE cellulose membrane is inserted into the slit.

Materials and Instruments

Restriction Endonuclease DNA Sample DNA Standard RNA
Ammonium Acetate DEAE High Salt Elution Buffer DEAE Low Salt Elution Buffer EDTA Ethanol Gel Loading Buffer NaOH Phenol Chloroform Sodium Acetate TE
Agarose Gel DEAE Cellulose Membrane Portable Long-Wave UV Lamp Water Bath

Move

I. Materials

1. Buffers and solutions

Ammonium acetate (10 mol/L)

DEAE high-salt elution buffer (50 mmol/L Tris-Cl (pH 8.0), 1 mol/L NaCI, 10 mmol/L EDTA (pH 8.0))

DEAE low-salt elution buffer (50 mmol/L Tris-Cl (pH 8.0), 0.15 mol/L NaCl, 10 mmol/L EDTA (pH 8.0))

EDTA ( 10 mmol/L, pH 8.0)

Ethanol

6X gel loading buffer

0.5 mol/L NaOH

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

Sodium Acetate (3 mol/L, pH 5.2)

TE ( pH 8.0)

2. Enzymes and buffers

Restriction endonucleases

3. Gel

Agarose gel containing 0.5 ug/ml EB

4. Nucleic acids and oligonucleotides

DNA samples

DNA standards

RNA, yeast transfer tRNA

5. Specialized equipment

DEAE cellulose membranes

Portable long-wave UV lamps

Water bath at 65°C

Methods

1. Digestion of a certain amount of DNA in order to harvest at least 100 ng of the target DNA. Separate the DNA fragments by agarose gel electrophoresis with appropriate concentration of 0.5 ug/ml EB. Localize the target band with a hand-held long-wave UV lamp.

2. Using a sharp blade or razor, cut a slit in the gel immediately in front of the target band, with both sides approximately 2 mm wider than the band.

3. Wearing gloves, cut a piece of DEAE cellulose membrane equal to the width of the slit and slightly deeper (1 mm) than the gel. After soaking in 10 mmol/L EDTA (pH 8.0) for 5 min at room temperature, the EDTA was replaced with 0.5 mol/L NaOH to activate the DEAE cellulose membrane. After another 5 min at room temperature, the membrane was rinsed 6 times with sterile water.

4. The membrane was inserted into the slit by propping up the incision wall with flat-tipped forceps. Remove the forceps and close the incision, being careful not to leave air bubbles.

5. Continue electrophoresis (<5 V/cm) until the DNA bands migrate onto the membrane. During electrophoresis, a handheld long-wave UV lamp (302 nm ) can be used for tracking.

6. When all the target DNA has left the gel and is collected on the membrane, cut off the current. Remove the membrane from the gel with flat-tip tweezers and rinse the membrane with 5 to 10 ml of DEAE Low Salt Buffer at room temperature to remove the agarose from the membrane.

7. Transfer the membrane to another microcentrifuge tube with sufficient amount of DEAE High Salt Elution Buffer so that the membrane is completely submerged. Gently crease or fold the membrane, but do not press. Cap the tube and incubate at 65 °C for 30 min.

8. During the elution of DNA from the membrane, image the gel and record the bands that have been isolated.

9. Transfer the liquid from step 7 to another microcentrifuge tube, add a sufficient amount of DEAE High Salt Elution Buffer, and incubate at 65 °C for 15 min. Combine the two portions of DEAE High Salt Solution.

10. The High Salt Elution Solution is extracted once with phenol:chloroform. Transfer the aqueous phase to another centrifuge tube. Add 0.2 times the volume of 10 mol/L ammonium acetate and 2 times the volume of 4°C ethanol and leave for 10 min at room temperature. Allow to stand at room temperature for 10 min. centrifuge in a microcentrifuge at room temperature for 10 min at maximum speed. carefully wash the precipitate with 70% ethanol. Open the tube for a few minutes to allow the ethanol to evaporate completely. Re-dissolve the DNA into 3-5 ul TE (pH 8.0).

11. If very pure DNA is required (e.g., for microinjection of fertilized mouse eggs or electroporation of cultured cells), the DNA can be reprecipitated in ethanol as follows.

(1) Suspend the DNA in 200 ul TE (pH 8.0), add 25 ul of 3 mol/L sodium acetate (pH 5.2), and reprecipitate the DNA in 2x the volume of ethanol. Reprecipitate the DNA with 2 times the volume of ethanol.

(2) Centrifuge the sample in a microcentrifuge at 4°C for 5-15 min at maximum speed to recover the DNA.

(3) Carefully rinse the precipitate with 70% ethanol. Open the centrifuge tube for a few minutes to allow the ethanol to evaporate completely. Dissolve the DNA in 3~5 ul TE (pH 8.0).

12. Characterize and quantify the DNA by gel electrophoresis. Mix a small amount (10-50 ng) of the final DNA fragment with 10 ul TE (pH 8.0) and add 2 ul of the desired gel loading buffer. Samples are added and electrophoresed on an agarose gel of appropriate concentration, using a known amount of the original DNA digested with the appropriate restriction enzyme as a reference standard and molecular quality standard. The isolated fragments should migrate in synchronization with the corresponding fragments in the restriction enzyme digestion product. Carefully examine the gel for the presence of weakly fluorescent bands. The presence of a weak fluorescent band indicates DNA contamination.

Caveat

1. The part cut off when cutting glue for recycling is as small as possible to improve the recycling efficiency.

2. Protective measures should be taken when exposed to ultraviolet radiation.

Common Problems

Materials

1. Buffers and solutions

Ammonium acetate (10 mol/L)

DEAE high salt elution buffer (50 mmol/L Tris-Cl (pH 8.0), 1 mol/L NaCI, 10 mmol/L EDTA (pH 8.0))

DEAE low salt elution buffer (50 mmol/L Tris-Cl (pH 8.0), 0.15 mol /L NaCl, 10 mmol/L EDTA (pH 8.0))

EDTA ( 10 mmol/L, pH 8.0)

Ethanol

6X gel loading buffer

0.5 mol/L NaOH

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

Sodium Acetate (3 mol/L, pH 5.2)

TE ( pH 8.0)

2. Enzymes and buffers

Restriction endonucleases

3. Gels

Agarose gel containing 0.5 ug/ml of EB

4. Nucleic acids and oligonucleotides

DNA samples

DNA standards

RNA, yeast transit tRNA

5. Specialized equipment

DEAE cellulose membrane

Portable long-wave UV lamp

65℃ water bath

Source "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.

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