DNA can be recovered from low melting point agarose gels by agarase digestion (Bumeister and lehrach 1989). In this method, the agarase enzyme hydrolyzes the agarose polymers to disaccharides. The DNA thus obtained is then purified by phenol extraction and ethanol precipitation. Because of its mildness, this method is particularly suitable for the recovery of high-molecular-quality DNA from pulsed-field agarose gels, but it is also effective for the recovery of small-molecule DNA from constant-field agarose gels. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Recovery of DNA from low melting point agarose gels (digestion with agarase)
Principle
DNA can be recovered from low melting point agarose gels by agarase digestion (Bumeister and lehrach 1989). In this method, the agarase enzyme hydrolyzes the agarose polymers to disaccharides. The DNA thus obtained is then purified by phenol extraction and ethanol precipitation. Because of its mildness, this method is particularly suitable for the recovery of high-molecular-quality DNA from pulsed-field agarose gels, but it is also effective for the recovery of small-molecule DNA from constant-field agarose gels.
Materials and Instruments
Agarase DNA Sample Move I. Materials For more product details, please visit Aladdin Scientific website.
Ethanol Ethidium bromide or SYBR Gold Staining Solution Gel Equilibration Buffer Bis Tris-Cl EDTA NaCl Equilibration Phenol TE
Boiled dialysis bag Microdialysis device UV lamp Water bath
1. Buffer and gemmellar solution
Ethanol
Ethidium bromide or SYBR Gold stains
Gel equilibration buffer (10 mmol/L Bis Tris-Cl ( pH 6.5), 5 mmol/L EDTA (pH 8.0), 0.1 mol/L NaCl)
NaCl ( 5 mol/L)
Equilibrium phenol (pH 8.0)
TE ( pH 8.0)
2. Enzyme and buffer
agarase
3. nucleic acids and oligonucleotides
DNA samples
4. Specialized equipment
Boiled dialysis bag
Microdialysis device (Life Technologies)
Portable Long Wave (302 nm) UV Lamps
Water baths with preset temperatures of 40°C and 65°C
II. Methods
1. Low melting point agarose gel was prepared, sampled and electrophoresed.
2. Cut off the bands containing the target DNA from the agarose gel, transfer to a microcentrifuge tube, add 20 times the volume of gel equilibrium buffer, and leave it at room temperature for 30 min.
3. Photograph the gel where the target DNA band is excised to record the excised DNA band.
4. Transfer the gel sections to a new microcentrifuge tube containing approximately the same volume of Gel Equilibration Buffer.
5. Incubate at 65°C for 10 min to melt the gel slices. Cool to 40°C and add 1 to 2 units of DNAase-free agarase per 200 μl of gel. incubate for 1 h at 40°C.
During this time, the agarose is cleaved to oligosaccharides or disaccharides. The DNA solution can be used directly for ligation, digestion, and transformation, if desired.
6. DNA purification and concentration
(1) Purification of small fragments of DNA (<20kb)
① Extract DNA solution twice with equilibrium phenol.
② After the second phenol extraction, transfer the aqueous phase to another new tube and add 2 times the volume of TE (pH 8.0).
(iii) Add 0.05 times the volume of 5 mol/L NaCl, followed by 2 times the volume of ethanol (the volume here refers to the final volume of DNA in step 6 (ii)). Pre-cool at 0℃ for 15 min, and centrifuge at max speed for 15 min at 4℃ in a microcentrifuge to collect the precipitate.
④ Carefully aspirate the ethanol and add 0.5 ml of 70% ethanol at room temperature. Mix well and centrifuge as described in step ③.
⑤ Pipette off the supernatant and open the tube. Place on the bench for a few minutes to evaporate the ethanol residue. Dissolve the DNA in an appropriate volume of TE (pH 8.0).
(2) Purification of large DNA fragments (>20kb)
① Transfer the agarase-digested sample to a dialysis bag, tie or seal the bag, and place it in a beaker or flask containing 100 ml of TE (pH 8.0).
Dialyze the sample at 4°C for several hours.