This method was first devised by Richaed Treisman (ICRF, London, UK) with reference to the work of Lis, who was the first to use polyethylene glycol (PEG) to isolate DNA of different sizes, and was widely used for the purification of plasmid DNA prepared by alkaline lysis. This experiment is based on the work of Lis, who was the first to use polyethylene glycol (PEG) to isolate DNA of different sizes.

Operation method

Experiment on purification of plasmid DNA by polyethylene glycol precipitation method

Principle

This method was first devised by Richaed Treisman (ICRF, London, UK) based on the work of Lis, who was the first to isolate DNA of different sizes using polyethylene glycol (PEG).The Treisman method has been widely used for the purification of plasmid DNA prepared by alkaline lysis.

Materials and Instruments

Crude Plasmid
Chloroform Ethanol Isopropanol LiCl PEG-MgCI2 Phenol Chloroform Sodium acetate TE
Sorvall SS-34 or equivalent rotary head Ice water bath

Move

I. Materials

1. Buffers and solutions

(1) Chloroform

(2) Ethanol

(3) Isopropyl alcohol

(4) LiCl ( 5 mol/L)

(5) _PEG-MgCI2 solution

(6) Phenol: chloroform (1:1, V/V)

(7) Sodium acetate (3 mol/L, pH 5.2 )

(8) TE ( pH 8.0 )

(9) TE ( pH 8.0) containing 20 μg/ml RNase A

2. Nucleic acids and oligonucleotides

Crude plasmids

3. Centrifuges and rotors

Sorvall SS-34 or its equivalent

4. Specialized equipment

Ice water bath

II. Methods

1. Transfer 3 ml of crude plasmid into a 15 ml Corex tube and cool to 0°C in an ice bath.

2. Add 3 ml of ice-cooled 5 mol/L LiCl, mix well, and centrifuge at 12000 g (Sorvall SS-34 turntable, 10000 r/min) for 10 min at 4 °C.

3. Transfer the supernatant to a 30 ml Corex tube, add an equal volume of isopropanol, mix well, and centrifuge at room temperature to recover nucleic acids at 12000 g (Sorvall SS-34 turntable, 10000 r/min) for 10 min.

4. Carefully pour off the supernatant and invert the tube so that the liquid runs dry. Wash the precipitate and tube walls with 70% ethanol at room temperature. Carefully pour off the ethanol and use a vacuum extractor to drain any remaining ethanol from the tube walls. Invert on a paper towel for several minutes so that no visible ethanol remains. However, the precipitate should be kept moist.

5. Dissolve the moistened nucleic acid precipitate with 500 μl of TE containing RNase A ( pH 8.0). Transfer the solution to a microcentrifuge tube and leave for 30 min at room temperature.

6. Extract the plasmid-RNase mixture once with phenol:chloroform and then once with chloroform.

7. Recover DNA by standard ethanol precipitation.

8. Dissolve the plasmid DNA precipitate in 1 ml of sterilized water and add 0.5 ml of _PEG-MgCl2 solution.

9. Allow to stand at room temperature for more than 10 min, then centrifuge at maximum speed for 20 min at room temperature in a microcentrifuge to recover the precipitated plasmid DNA.

10. The precipitate is resuspended with 0.5 ml of 70% ethanol to remove traces of PEG, and centrifuged in a microcentrifuge at maximum speed for 5 min to recover nucleic acids.

11. Aspirate off the ethanol and repeat step 10. after the second wash, leave the tubes on a rack for 10-20 min to allow the ethanol to evaporate.

12. Dissolve the wetted plasmid precipitate in 500 μl TE (pH 8.0). Dilute 1:100 with TE (pH 8.0) and measure _OD260 and calculate the concentration of plasmid DNA. Calculate the concentration of plasmid DNA as _1OD260 = 50 μg plasmid DNA/ml.

13. Dispense the DNA and store at -20℃.

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