This method is a modification of the Bowtell method (1987), which allows for the simultaneous preparation of DNA from many different cell or tissue samples.The main steps in this protocol consist of precipitating the DNA at the interface between the cell lysate and ethanol, followed by wrapping the precipitated DNA around a Shepherd's hook. The DNA wrapped around the hook is transferred from the ethanol solution to be dissolved in the liquid buffer of choice. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Isolation of DNA from mammalian cells by the winding method
Principle
This method is a modification of the Bowtell method (1987), which allows for the simultaneous preparation of DNA from many different cell or tissue samples.The main steps in this protocol consist of precipitating the DNA at the interface between the cell lysate and ethanol, followed by wrapping the precipitated DNA around a Shepherd's hook. The DNA wrapped around the hook is spun out of the ethanol solution and dissolved in the liquid buffer of choice. This method of collecting high molecular mass DNA precipitates was first used in the 1930s. Small fragments of DNA and RNA do not integrate efficiently into gel-like wraps. These DNAs are often too small (~80 kb) to be used in the construction of genomic DNA libraries, but they work well for Southern hybridization and polymerase chain reaction, and can be partially digested by restriction endonucleases for the construction of size-graded libraries. 1 ml of cultured aneuploid mammalian cells (2.0X107, e.g., HeLa cells) produces 100 μg of DNA. g DNA.
Materials and Instruments
λ Linear monomers and multiplexes of phages Mammalian cells, fresh tissue or blood samples Move I. Materials For more product details, please visit Aladdin Scientific website.
Cell lysate Ethanol TE
Pulsed electric field gels or 0.6% agarose gels Kimwipe Absorbent paper Polypropylene tubes Vibrating platforms Shepherd's hooks Wide-mouth pipettes
1. Buffers and solutions
Cell lysis solution (6 mol/L guanidine hydrochloride, 0.1 mol/L sodium acetate (pH 5.5))
Ethanol (room temperature)
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
Kimwipe absorbent paper
Polypropylene tube (50 ml)
Oscillating platform
Shepherd's hook (an alternative to dialysis)
Wide-mouth pipettes (0.3 cm diameter at opening)
5. Cells and tissues
Mammalian cells, fresh tissues or blood samples in monolayer or suspension culture
II.
1. Prepare lysates of cell suspensions (or frozen cell powders).
2. Prepare a cell lysate using one of the following two methods.
(1) Lysis of cells from suspension culture solution
① Transfer the cell suspension to a disposable polypropylene centrifuge tube.
① Transfer the cell suspension to a disposable polypropylene centrifuge tube. ② Add 7.5 times the volume of cell lysate.
(2) Cell lysis from tissue
① Add frozen cell powder little by little to a beaker containing about 7.5 times the volume of cell lysate, dispersing it on the surface of the lysate and shaking the beaker to submerge the powder.
② When all materials are dissolved, transfer the solution to a centrifuge tube.
3. Close the cap of the centrifuge tube and incubate for 1 h at room temperature on a shaker.
4. Add 18 ml of ethanol at room temperature to a series of disposable 50 ml polypropylene centrifuge tubes. Use a wide mouth pipette to carefully keep the cell suspension below the ethanol.
5. Slowly agitate the interface between the cell lysate and ethanol with a Shepherd's hook to recover DNA, which will adhere to the hook and form a gel. Continue stirring until the ethanol and water phases are completely mixed.
6. Transfer the DNA-adhered Shepherd's hooks to another polypropylene tube containing 5 ml of room temperature ethanol. Immerse the DNA in ethanol until all samples are complete.
7. Remove each Shepherd's hook with DNA adhering to it, allowing the ethanol to drip dry as much as possible. At this point, the DNA has contracted into tightly packed dehydrated clumps. Most of the free ethanol is usually removed by capillary action by contacting the U-shaped end of the hook with Kimwipe absorbent paper. When the ethanol on the DNA has not completely evaporated, the hooks are transferred to a new polypropylene tube containing 5 ml of ethanol at room temperature.
8. When all samples have been processed, remove as much ethanol as possible.
9. Transfer each pipette to a new polypropylene tube containing 1 mI TE buffer (pH 8.0). Leave overnight at 4°C to rehydrate the DNA.
10. During the hydration process, the DNA will be highly gelatinous but still adhere to the pipette. Using another new Shepherd's hook as a spatula, gently scrape the DNA precipitate off the pipette. Discard the pipette and let the DNA precipitate float in the TE. Cap the tube and incubate at 4°C on a shaker until the DNA precipitate is completely dissolved (~24-48 h).
11. Analyze on pulse gel electrophoresis or 6% agarose gel.