Pulsed-field gel electrophoresis with clamped uniform electric field
Pulsed-field gel electrophoresis with clamped uniform electric field
In clamped homogeneous electric field (CHEF) gel electrophoresis, the electric field is generated by multiple electrodes. These electrodes are arranged in a quadrilateral or hexagonal shape around a horizontal gel, and their potentials are clamped at a top-set level (Chuetal. 1986; Vollrath and Davis 1987; Chu 1990a). The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.
Operation method
Pulsed-field gel electrophoresis with clamped uniform electric field
Principle
In clamped homogeneous electric field (CHEF) gel electrophoresis, the electric field is generated by multiple electrodes. These electrodes are arranged in a quadrilateral or hexagonal shape around a horizontal gel, and their potentials are clamped at a top-set level (Chuetal. 1986; Vollrath and Davis 1987; Chu 1990a). The electric fields generated when the perimeter is quadrilateral are at right angles to each other; those generated by hexagonal shapes are at 60° or 120°, depending on the position of the gel and the polarity of the electrodes.
Materials and Instruments
DNA Size Standard Target Genomic DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Denaturing Buffer Gel Electrophoresis Buffer
High Quality Agarose CHEF Gel Equipment Circulating Water Bath Water Bath
1. Buffers and solutions
Denaturing buffer (0.5 N NaOH, 1.5 mol/L NaCI)
Ethidium bromide (1 μg/ml) or appropriate dilutions of SYBR Gold
0.5X TBE gel electrophoresis buffer
2. Gel
High quality agarose
3. Nucleic acids and oligonucleotides
DNA size standard
Target genomic DNA
4. Specialized equipment
CHEF Gel Device
Circulating water bath
14℃ water bath
II. Methods
CHEF Separation of DNA fragments
1. Prepare an agarose gel of appropriate concentration with 0.5 X TBE buffer and cast. Monitor with a bubble level to ensure that the tray is perfectly level on the bench. Allow the gel to harden for 1 h at room temperature.
2. Place the gel in the CHEF apparatus, add enough 0.5 X TBE to just cover the gel, and cool the remaining buffer to 14 °C.
3. Prepare an agarose plug containing the target DNA and perform restriction enzyme digestion. Prepare and embed DNA size standards.
4. Gently place the plugs into microcentrifuge tubes, add 200 μl of 0.5 x TBE to each tube, and incubate for 15 min at room temperature.
5. Digested and washed DNA plugs are individually embedded in gel spiking wells and the gel plugs are sealed with melted gelatinizing agarose.
6. Allow the blocked gel to harden for approximately 5 min, add additional 0.5X TBE buffer (previously cooled to 14°C in Step 2) to completely cover the gel.
7. Start a buffer cycle and electrophoresis under power conditions.
8. After electrophoresis, stain the gel with ethidium bromide or appropriate dilution of SYBR Gold at room temperature for 30 min. destain in water for 30 min, and photograph under UV light.
DNA denaturation is transferred to a nylon membrane.
9. After the photo, the gel was incubated in 250 ml denaturing buffer with slight shaking for 30 min, changed the denaturing buffer and incubated for another 30 min.
10. Transfer DNA directly to the nylon membrane in denaturing buffer using capillary blotting.
11. After transfer, bake at 80°C for 2 h or crosslink with UV or microwave to fix the DNA on the nylon membrane.
12. Pre-hybridization and hybridization with labeled probes in buffer containing formamide.