This procedure is adapted from a method first described by Daryl Stafford and colleagues (Blin and Stanfford 1976). This method should be used when large quantities of mammalian DNA are required, such as for Somhern hybridization or for the construction of genomic libraries with phage λ vectors. Approximately 200 μg of mammalian DNA, 100-150 kb in length, can be obtained from 5X107 cultured aneuploid cells (e.g. HeLa cells).
Operation method
Isolation of high molecular mass DNA from mammalian cells using proteinase K and phenol
Principle
This procedure is adapted from a method first described by Daryl Stafford and colleagues (Blin and Stanfford 1976). This method should be used when large quantities of mammalian DNA are required, such as for Somhern hybridization or for the construction of genomic libraries with phage λ vectors. Approximately 200 μg of mammalian DNA, 100-150 kb in length, can be obtained from 5X107 cultured aneuploid cells (e.g. HeLa cells).
Materials and Instruments
Proteinase K Phage λ DNA Mammalian cells, fresh tissue, or blood samples Move I. Materials For more product details, please visit Aladdin Scientific website.
Ammonium acetate Dialysis buffer Ethanol Lysis buffer Phenol TE Tris-buffered salt solution
Pulsed electric field gels or conventional horizontal 0.6% agarose gels Sorvall centrifuges and H1000B, SS-34 Turning heads Tip-cut yellow tips Dialysis tubing clamps Oscillating platforms or dialysis tubing Shepherd's hooks Spectrophotometers or fluorometers Rotators Vacuum dryers Water baths Wide-mouth pipettes
1. Buffers and solutions
Ammonium acetate (10 mol/L) (an alternative to dialysis, step poly 9)
Dialysis buffer (an alternative to ethanol precipitation, step 9) (50 mmol/L Tris-Cl (pH 8.0), 10 mmol/L EDTA (pH 8.0), prepare 4 parts of 4 L dialysate and store at 4°C)
Ethanol (an alternative to dialysis, step 9)
Lysis buffer (10 mmol/L Tris-Cl (pH 8.0), 0.1 mol/L EDTA (pH 8.0), 0.5% (m/V) SDS, 20 μg/mg tryptic RNase without DNase)
Phenol, equilibrated with 0.5 mol/L Tris-Cl ( pH 8.0)
TE (pH 8.0)
Tris-buffered salt solution (TBS)
2. Enzyme and buffer
Protease K ( 20 mg/ml)
3. gel
Pulsed electric field gel or conventional horizontal 0.6% agarose gels
4. nucleic acids and oligonucleotides
Intact phage λ DNA
5. Centrifuges and rotors
Sorvall centrifuge and H1000B, SS-34 Turning head (or other equivalent)
6. Specialized equipment
Yellow tip for tip cutting
Dialysis Tube Clamps
Oscillating Platform or Dialysis Tubing
Shepherd's hook (an alternative to dialysis)
Spectrophotometer or fluorometer
Rotator
Vacuum dryer
50°C Water Bath
Wide mouth pipette (0.3 cm diameter at opening)
7. Cells and tissues
Mammalian cells, fresh tissues or blood samples in monolayer or suspension culture
II.
Below are 4 different versions of lysing different types of cell and tissue samples Step 1. Depending on the material under study, choose the appropriate method for lysis followed by Step 2.
1. Lysis of monolayer cultured cells 
It is best to make 10-12 petri dishes at a time and store the rest in an incubator, removing them before use.
(1) Remove a batch of dishes full of monolayer cells from the incubator, quickly aspirate the culture solution and wash it twice with ice-cold TBS. Carefully add 10 ml of TBS, swirl gently for a few seconds and pour the solution into a 2 L beaker. Add 10 ml of ice-cold TBS and place on ice. Repeat the process for the entire batch of cells.
(2) Pour the TBS solution into a 2 L beaker and aspirate the remaining solution. Add 1 ml of fresh ice-cold TBS and place the dish on ice. Repeat the process for the whole batch of cells.
(3) Use a rubber spatula to scrape the cells into 1 ml of TBS. Use a Pasteur pipette to transfer the cell suspension to a centrifuge tube on ice. Rinse the petri dish with 0.5 ml of ice-cold TBS and incorporate the cell suspension in the centrifuge tube. Repeat this process to make the entire batch of cells.
(4) Centrifuge at 1500 g (approximately 2700 r/min) for 10 min to collect the cells.
(5) Resuspend the cells in 5-10 times the volume of ice-cold TBS and centrifuge again.
(6) Resuspend the cells in TE (pH 8.0) to a concentration of 5X107 cells/ml and transfer to a triangular flask.
(7) Add 10 ml of lysis buffer per ml of cell suspension and incubate at 37℃ for 1 h, and proceed to step 2 immediately.
2. Lysis of suspended cultured cells 
(1) Transfer the cells to a centrifuge tube and centrifuge at 1500 g (Sorvall H100B head, 2700 r/min) at 4°C for 10 min to collect the cells. Aspirate off the supernatant.
(2) Resuspend the cells in 1x volume of ice-cold TBS and centrifuge again. Aspirate the supernatant, carefully resuspend the cells again in ice-cold TBS and centrifuge to collect the cells.
(3) Remove the supernatant and carefully resuspend the cells in TE (pH 8.0) to 5X107 cells/ml, transfer the supernatant to a triangular flask.
(4) Add 10 ml of lysis buffer to each ml of cell suspension and incubate at 37℃ for 1 h, proceed to step 2 immediately.
3. Lysis of tissue samples
Since tissues usually contain a large amount of fibrous material, it is difficult to obtain high yields of genomic DNA from them, and pulverizing the tissue prior to lysis greatly improves the extraction efficiency. Large amounts of fresh tissue (>1 g) can be pulverized with a Waring blender. 
Grinding of Tissue Samples
(1) Place 1 g of freshly cut tissue sample in a stainless steel cup containing a liquid nitrogen Waring stirrer and pulverize the tissue at maximum speed.
(2) When the liquid nitrogen evaporates, add the tissue powder little by little to a beaker containing 10 times the volume (m/V) of lysate, dispersing it on the surface of the lysate and shaking the beaker to submerge the powder.
(3) When the powder is completely dispersed in the solution, transfer the suspension to a 50 ml centrifuge tube and incubate at 37°C for 1 h. Immediately proceed to step 2.
4. Lysis of freshly drawn or frozen blood cells 
(1) Collect blood cells from freshly drawn or frozen samples (human blood should be drawn by a skilled blood collector in a sterile environment)
Collecting Blood Cells from Fresh Blood Samples
① Collect 20 ml of fresh blood in a tube containing 3.5 ml of citrate dextrose solution B (ACD) or EDTA.
② Transfer the blood sample to a centrifuge tube and centrifuge at 1300 g (Sorvall H100B turntable, 2500 r/min) at 4°C for 15 min.
(iii) Aspirate the supernatant. The yellowish layer was carefully transferred to a new tube with a Pasteur pipette and centrifuged again. Discard the erythrocyte precipitate.
The yellowish layer is a broad band of leukocytes of uneven density.
④ Resuspend the yellowish layer in 15 ml of lysis buffer and incubate at 37°C for 1 h, then proceed to step 2.
Collection of blood cells from frozen blood samples
① Collect 20 ml of fresh blood in a tube containing 3.5 ml of citrate dextrose solution B (ACD) or EDTA.
Blood samples may be stored at 0°C for several days and stored indefinitely at -70°C.
② Thaw blood samples in a room temperature water bath and transfer to a centrifuge tube with an equal volume of phosphate buffer.
(iii) Centrifuge at 3500 g (Sorvall SS-34 head, 5400 r/min) for 15 min at room temperature.
④ The supernatant containing lysed erythrocytes was aspirated. The supernatant containing lysed erythrocytes was aspirated and the precipitate was resuspended in 15 ml of lysis buffer. The sample was incubated at 37°C for 1 h, and then proceeded to step 2.
Treat the cell lysate with proteinase K and phenol.
2. Transfer the lysate to one or more centrifuge tubes with Sorvall SS-34 turntables, not exceeding 1/3 of the volume of the lysate.
3. add Proteinase K (20 mg/ml) to a final concentration of 100 μg/ml. use a glass rod to gently mix the enzyme into the viscous cell lysate.
4. Place the cell lysate in a 50 °C water bath for 3 h, rotating the viscous solution from time to time.
5. Cool the solution to room temperature and add an equal volume of phenol equilibrated with 0.1 moI/L Tris-Cl (pH 8.0). Place the centrifuge tube on a spinner and slowly invert the tube for 10 min to gently mix the two phases. If the two phases do not form an emulsion by this time, the tube is placed on the spinner for 1 h. The tube is then turned upside down for 10 min to gently mix the two phases.
6. Separate the two phases by centrifugation at 5000 g (Sorvall SS-34 turntable, 6500 r/min) for 15 min at room temperature.
7. Transfer the viscous aqueous phase to another centrifuge tube using a wide-mouth pipette (outlet diameter 0.3 cm).
8. Extract twice more with phenol and collect the aqueous phase.
9. Isolate the DNA by one of the following two methods.
(1) Isolation of DNA with a size of 150 to 200 kb.
① Transfer the aqueous phase to a dialysis bag, which is sealed at the top with a dialysis tubing clip, leaving space in the bag to increase the volume of the sample by 1.5 to 2 times the volume of the sample.
② Dialyze the sample in 4 L of dialysis buffer at 4 ℃, and change the buffer 3 times at 6 h intervals.
Due to the high viscosity of DNA, dialysis usually takes more than 24 hours to complete.
(2) Isolation of DNA with an average size of 100~150 kb
① After the third extraction with phenol, transfer the aqueous phase to another centrifuge tube with 0.2 times the volume of 10 mol/L ammonium acetate and 2 times the volume of ethanol, and spin the tube until the solution is thoroughly mixed.
A precipitate of ② DNA was formed. The DNA precipitate is removed from the ethanol solution using a Shepherd's hook (Pasteur pipette with the end sealed and pulled into a U-shape), leaving the contaminating oligonucleotides in place.
(iii) If the DNA precipitate becomes fragmented, collect the precipitate by centrifugation at 5000 g for 5 min at room temperature without a Shepherd's hook.
④ Wash the DNA precipitate twice with 70% ethanol and collect the DNA by centrifugation as in step ③.
⑤ Aspirate off the residual ethanol with a vacuum pump as much as possible. Place the DNA precipitate in an open tube at room temperature until visible traces of ethanol have evaporated.
Do not allow the DNA precipitate to dry completely or the DNA will be extremely difficult to dissolve.
(vi) Add 1 ml of TE for every 0.1 ml of cells (step 1) and place on a shaker platform and shake gently at 4°C for 12-24 h until the DNA is completely dissolved. Store the DNA solution at 4°C.
10. Determine the concentration of DNA.
11. Analyze the quality of the prepared high molecular mass DNA by pulse gel electrophoresis or conventional 0.6% agarose gel electrophoresis. Use λDNA monomers or linear multiplexes as molecular mass markers. 
