Pulsed-field gel electrophoresis with transverse alternating fields
Pulsed-field gel electrophoresis with transverse alternating fields
Gardiner et al. (1986; Gardiner and Patterson 1989) used a gel electrophoresis device with two sets of platinum wire electrodes placed on either side of a vertical gel to separate large DNA fragments. In this type of transverse alternating field electrophoresis (TAFE), DNA moves first to the anode of one set of electrodes and then to the anode of the other set of electrodes when the electric field is switched. This experiment is from "Molecular Cloning Laboratory Guide, Third Edition", translated by Huang Peitang et al.
Operation method
Pulsed-field gel electrophoresis with transverse alternating fields
Principle
Gardiner et al. (1986; Gardiner and Patterson 1989) used a gel electrophoresis device with two sets of platinum wire electrodes placed on either side of a vertical gel to separate large DNA fragments. In this type of transverse alternating field electrophoresis (TAFE), when the field is switched, the DNA moves first to the anode of one set and then to the anode of the other set.
Materials and Instruments
DNA Molecular Quality Standards Target Genomic DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Denaturing Buffer TAFE Gel Electrophoresis Buffer TE
High Quality Agarose Circulating Water Bath TAFE Gel Equipment Water Baths
1. Buffers and solutions
Denaturing buffer (0.5 N NaOH, 1.5 mol/L NaCl)
TAFE gel electrophoresis buffer containing 0.5 μg/ml ethidium bromide or appropriate dilution of SYBR Gold
TAFE gel electrophoresis buffer (20 mmol/L Tris-acetic acid (pH 8.2), 0.5 mmol/L EDTA)
TE ( pH 8.0)
2. Gel
High quality agarose
3. Nucleic acids and oligonucleotides
DNA Molecular Quality Standards
Target genomic DNA
4. Specialized equipment
Circulating water bath
TAFE gel equipment
Set up a 14°C water bath
II. Methods
TAFE Separation of DNA Fragments
1. Make a 1% agarose gel in 1X TAFE buffer without ethidium bromide and allow it to set up. The buffer used to make the gel is the same as the buffer used to fill the electrophoresis tank.
2. Prepare an agarose bolus containing the target DNA and perform restriction enzyme digestion. Prepare and embed DNA standards.
3. Rinse all plugs in 10x volume of TE (pH 8.0) for 30 min with two changes of solution.
4. Digested and washed DNA plugs are embedded into gel wells and the plugs are sealed with 1% agarose dissolved in 1X TAFE buffer.
5. Place the gel into a TAFE gel tank containing 1x TAFE buffer, which has been cooled at 14℃.
6. Turn on the power and electrophoresis for 30 min at a constant current of 170~180 mA with a 4s pulse; this treatment allows the DNA to be rapidly incorporated into the gel. Thereafter, reduce the current input to 150 mA, set the pulse time to the optimal selection for DNA isolation, and continue electrophoresis for 12~18 h.
7. Disconnect the power supply, remove the gel and stain it in 1x TAFE buffer containing 0.5 μg/ml ethidium bromide or appropriate dilution of SYBR Gold. Photograph under UV light. 
DNA denatured and transferred to a nylon membrane
8. Wash the stained gel twice with water. After two washes, pour off the water and add Denaturation Buffer and incubate for 30 min with gentle shaking. Replace the Denaturation Buffer and incubate for another 30 min.
9. Transfer DNA directly to the nylon membrane in denaturing buffer using capillary blotting.
10. After transfer, bake at 80°C for 2 h or crosslink with UV or microwave to fix the DNA on the nylon membrane.
11. Pre-hybridization and hybridization with labeled probes in buffer containing formamide. 