Alkaline agarose gel electrophoresis is performed at high pH, which causes the loss of a proton from thymine and guanine residues, thus preventing the formation of hydrogen bonds between their respective paired bases, adenine and cytosine. The denatured DNA remains in a single-stranded state and swims in an alkaline agarose gel depending on its molecular size. Other denaturants such as formamide and urea tend to give poorer results because they cause the agarose to rubberize. The source of this experiment is "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Alkaline agarose gel electrophoresis
Principle
Alkaline agarose gel electrophoresis is performed at high pH, which causes the loss of a proton from thymine and guanine residues, thus preventing the formation of hydrogen bonds between their respective paired bases, adenine and cytosine. The denatured DNA remains in a single-stranded state and swims in an alkaline agarose gel depending on its molecular size. Other denaturants such as formamide and urea tend to give poorer results because they cause the agarose to rubberize.
Materials and Instruments
DNA Sample Move I. Materials For more product details, please visit Aladdin Scientific website.
Agarose Alkaline agarose electrophoresis buffer Alkaline gel loading buffer Ethanol Ethidium bromide or SYBR Gold staining solution Alkaline agarose gel neutralization solution Sodium acetate TAE electrophoresis buffer
Glass plates Water bath
1. Buffers and solutions
Agarose
10X alkaline agarose electrophoresis buffer (500 mmol/L NaOH, 10 mmol/L EDTA)
6X Alkaline gel loading buffer (300 mmol/L NaOH, 6 mmol/L EDTA, 18% (m/V) Ficoll-400 ( Pharmacia Corporation), 0.15% (m/V) bromocresol green, 0.25% (m/V) xylene cyanide)
Ethanol
Ethidium bromide or SYBR Gold staining solution
Alkaline agarose gel neutralization solution (1 mol/L Tris-Cl (pH 7.6), 1.5 mol/L NaCl)
Sodium acetate (3 mol/L pH 5.2)
1X TAE electrophoresis buffer
2. Nucleic acids and oligonucleotides
DNA samples (usually radiolabeled)
3. Specialized equipment
Glass plates
Water bath with pre-set temperature of 55°C 
II. Methods
1. Prepare agarose solution in a triangular flask or glass bottle by adding exact amount of agarose powder and quantitative amount of water.
2. Gently stuff the neck of the flask with Kimwipes paper. If a glass flask is used, the stopper must be loosened. Heat the suspension in a boiling water bath or microwave until the agarose is dissolved.
3. Allow the clear solution to cool to 55 °C. Add 0.1 times the volume of 10 µg/kg of agarose. Add 0.1 times the volume of 10X Alkaline Gel Electrophoresis Buffer and quickly fill the gel. When the gel has completely solidified, place it in the electrophoresis tank and add freshly prepared 1X Electrophoresis Buffer until the gel is completely covered.
4. Collect the DNA samples by conventional ethanol precipitation method. Add 10-20 μl of 1X gel buffer to dissolve the precipitate, and then add 0.2 times the volume of 6X alkaline gel loading buffer.
5. Add the DNA sample dissolved in 6X Alkaline Carrier Buffer to the spiking wells. Start electrophoresis at <3.5 V/cm and turn off the power when the bromocresol green has migrated 0.5-1 cm. Place a glass plate on top of the gel and continue electrophoresis until the dye has migrated nearly 2/3 of the gel length.
6. Depending on the purpose of the experiment, treat the gel according to one of the procedures described below.
(1) Southern hybridization
① Soak the gel in a neutralizing solution at room temperature for 45 min. transfer the DNA onto an uncharged nitrocellulose or nylon membrane.
② Hybridize the solidified DNA on the membrane with the appropriate labeling probe.
(2) Staining
① Soak the gel in neutralizing solution for 45 min at room temperature.
② Stain the neutralized gel with 1X TAE or SYBR Gold containing 0.5 μg/ml ethidium bromide.
Radiation autochromatography of wet gels: Follow one of the methods described in the column below. 
