This protocol is a modified version of the method described by Kupiec et al. (1987), which consists of digestion of cells and tissues with proteinase K, separation of the DNA-protein complexes with a high concentration of formamide, and removal of the protease and organic solvents by adequate dialysis of the chromatin in a pouch of hot cotton wool. Formamide is an ionizing solvent that separates protein-DNA complexes and denatures and releases proteins, but it does not significantly affect the activity of Proteinase K. This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

Operation method

Isolation of high molecular mass DNA from mammalian cells using formamide

Principle

This protocol is a modified version of the method described by Kupiec et al. (1987), which consists of digestion of cells and tissues with proteinase K, separation of the DNA-protein complexes with a high concentration of formamide, and removal of the protease and organic solvents by adequate dialysis of the chromatin in a pouch of hot cotton wool. Formamide is an ionizing solvent that separates protein-DNA complexes and denatures and releases proteins, but it does not significantly affect the activity of Proteinase K. The procedure is not intended to be used as a solvent. The genomic DNA prepared by this procedure is very large (7200 kb) and is suitable for library construction with high packaging capacity vectors and for analysis of large DNA fragments by pulsed gel electrophoresis.This method has two drawbacks: it requires more time than the other methods, and the final concentration of the prepared DNA is rather low (about 10 μg/ml). Approximately 1 mg of high-molecular-quality DNA can be prepared from 1X108 cultured aneuploid mammalian cells (e.g., HeLa cells).

Materials and Instruments

λ Linear monomers and multiplexes of phages Mammalian cells Fresh tissue Blood samples
Dialysis buffer Formamide denaturing buffer TE
Pulsed electric field gels or 0.6% agarose gels Stirrers with stainless steel cups Hot cotton gel bags Yellow tips for tip removal Dialysis tubing clips Glass rods Water baths or incubators

Move

I. Materials

1. Buffers and solutions

Dialysis buffer 1 (20 mmol/L Tris-Cl (pH 8.0), 0.1 moI/L NaCl, 10 mmol/L EDTA (pH 8.0), prepare 6 L of dialysis buffer 1 and store at 4°C.)

Dialysis Buffer 2 (10 mmol/L Tris-Cl (pH 8.0), 10 mmol/L NaCl, 0.5 mmol/L EDTA (pH 8.0), prepare 6 L of Dialysis Buffer 2 and store at 4°C.)

Formamide Denaturing Buffer (20 mmol/L Tris-Cl ( pH 8.0), 0.8 mol/L NaCI, 80% (V/V) formamide)

TE ( pH 8.0)

2. Gel

Pulsed electric field gel or 0.6% agarose gel

3. nucleic acids and oligonucleotides

λ Linear monomers and multiplexes of phages

4. Specialized equipment

Stirrer with stainless steel cup (for tissue)

Firewool plastic bags

Yellow tip for tip removal

Dialysis Tube Clips

Glass Rods

Water bath or incubator pre-set at 15°C

Pre-set in a water bath at 50°C

5. Cells and tissues

Mammalian cells in monolayer or suspension culture, or fresh tissue, or blood samples

II. Methods

1. Prepare lysates of cell suspensions (or frozen cell powders) using steps 1~4 of Scheme 1.

2. Cool the solution containing the lysed cells and the lysis buffer to 15°C. Add 1.25 ml of formamide denaturing buffer to every 1 ml of cell lysate and mix the solution gently with a glass rod. Allow the solution to stand for 12 h at 15°C.

3. Pour the viscous solution into one or more Fiberfoam bags. Seal the openings of the bags with dialysis tubing clamps and dialyze for 45 min at 4°C in 2 L of Lysis Buffer 1. Replace with Fresh Buffer 1 and continue dialyzing for at least 4 h. Replace with 2 L of Fresh Dialysis Buffer 1 and dialyze for an additional 4 h. Dialyze the DNA in 2 L of Fresh Dialysis Buffer 2 three times for at least 4 h each time.

4. Determine the concentration of DNA.

5. Pulse gel electrophoresis or conventional 0.6% agarose gel electrophoresis. Use λDNA monomers or linear multiplexes as molecular mass markers. The genomic DNA size will be over 200 kb.

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