Hydroxyethyl-modified agarose reduces the number of hydrogen bonds between chains and can melt and solidify at lower temperatures than standard agarose. The extent to which this substitution occurs on the polysaccharide chains determines the exact temperature at which the agarose melts and solidifies. This property forms the basis for the recovery and manipulation of DNA from gels (Wielander 1979; Parker and Seed, 1980). 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 (organic solvent extraction)
Principle
Hydroxyethyl-modified agarose reduces the number of hydrogen bonds between chains and can melt and solidify at lower temperatures than standard agarose. The extent to which this substitution occurs on the polysaccharide chains determines the exact temperature at which the agarose melts and solidifies. This property forms the basis for the recovery and manipulation of DNA from gels (Wielander 1979; Parker and Seed, 1980).
Materials and Instruments
DNA Sample Move I. Materials For more product details, please visit Aladdin Scientific website.
Ammonium acetate Chloroform Ethanol Ethidium bromide or SYBR Gold Staining solution Loading buffer LMT Elution buffer Tris-HCl EDTA Phenol Chloroform Equilibrium saturated phenol TAE Electrophoresis buffer TE
Low melting point agarose preparative gel Sorvall SS-3 or equivalent rotor UV lamp Water bath
1. Buffers and solutions
Ammonium acetate (10 mol/L)
Chloroform
Ethanol
Ethidium Bromide or SYBR Gold Stain
6X loading buffer
LMT elution buffer (20 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA (pH 8,0))
Phenol: chloroform (1:1, V/V)
Equilibrium saturated phenol (pH 8.0)
1X TAE Electrophoresis Buffer
TE ( pH 8.0)
2. Gel
Preparation of gels with low melting point agarose
3. Nucleic acids and oligonucleotides
DNA samples
4. centrifuges and rotors
Sorvall SS-3 or equivalent rotors
5. Specialized equipment
Portable long wavelength (302 nm) UV lamps
Water bath with preset temperature of 65°C
II. Methods
1. A piece of low melting point agarose gel containing appropriate concentration of TAE electrophoresis buffer was filled with 1X TAE electrophoresis buffer.
TAE was chosen over TBE for several reasons. The main reason is that the borate ions in TBE inhibit the ligation reaction and can interfere with the purification of DNA fragments eluted from the glass beads.
2. After the gel has cooled to room temperature, transfer the gel, along with the supporting glass plate, to the horizontal surface of the gel box.
The gel can also be placed in a cold room to ensure adequate gelation.
3. Mix the DNA sample with loading buffer and inject into the spiking wells for electrophoresis at 3~6 V/cm.
DNA of a particular molecular mass size swims faster in low melting point agarose gels than in conventional agarose gels. Because of this, the voltage applied to low melting point agarose should be lower than that of conventional agarose gels.
4. If desired, stain with ethidium bromide or SYBR Gold. Determine the exact location of the desired band with a hand-held long-wave (302 nm) UV lamp.
5. Cut the agarose gel section containing the desired band with a sharp blade or razor and transfer to a clean, single-use plastic tube.
Try to minimize the volume of the cut agarose slice to reduce the amount of DNA contamination by the inhibitor.
6. After the bands are cut, photograph them to obtain a record of the cut bands.
7. Add approximately 5 times the volume of LMT Elution Buffer to the agarose sections. Cap the tube and incubate at 65°C for 5 min to melt the gel.
8. Allow the gel to cool to room temperature, add an equal volume of equilibrium phenol, and mix the mixture for 20 s. Centrifuge at 4000 g (Sorvail SS-34 rotor 5800 r/min) for 10 min at 20 °C to recover the aqueous phase.
9. The aqueous phase was extracted once more with an equal volume of phenol: chloroform and chloroform.
10. Transfer the aqueous phase to a new centrifuge tube. Add 0.2 times the volume of 10 mol/L ammonium acetate and 2 times the volume of anhydrous ethanol at 4℃. The mixture was allowed to stand at room temperature for 10 min, then centrifuged at 5000 g (Sorvall SS-34 rotor equivalent to 6500 r/min) for 20 min at 4°C, and the DNA was recovered by precipitation.
11. Wash the precipitate with 70% ethanol and dissolve in an appropriate volume of TE (pH 8.0). 