DNA preparation by pulsed-field gel electrophoresis (isolation of mammalian cell and tissue DNA)
DNA preparation by pulsed-field gel electrophoresis (isolation of mammalian cell and tissue DNA)
This protocol describes methods for preparing DNA samples from cultured cells and tissues. Leukocytes of patient or animal origin can also be used as high molecular mass DNA samples for PFGE. If necessary, DNA can also be prepared from isolated nuclei of mammalian cells. Experience has shown that there is no advantage to preparing DNA from the nucleus because DNA prepared from intact cells is just as easy to digest. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Peitang Huang et al.
Operation method
DNA preparation by pulsed-field gel electrophoresis (isolation of mammalian cell and tissue DNA)
Principle
This protocol describes methods for preparing DNA samples from cultured cells and tissues. Leukocytes of patient or animal origin can also be used as high molecular mass DNA samples for PFGE. If necessary, DNA can also be prepared from isolated nuclei of mammalian cells. Experience has shown that there is no advantage to preparing DNA from the nucleus because DNA prepared from intact cells is just as easy to digest.
Materials and Instruments
Cell or tissue samples Move I. Materials For more product details, please visit Aladdin Scientific website.
EDTA L buffer Phosphate buffer saline Erythrocyte lysis buffer TE
Low melting point agarose Sorvall SS-34 or equivalent rotary head Gauze Glass homogenizer Cell counting plates LeucoPrep cell separation tubes Mortar and pestle Water bath
1. Buffers and solutions
EDTA ( 0.5 mol/L, pH 8.0)
L Buffer (0.1 mol/L EDTA (pH 8.0), 0.01 mol/L Tris-Cl (pH 7.6), 0.02 mol/L NaCl, stored at 4°C)
L buffer containing proteinase K and sodium dodecyl sarcosinate (Sarkosyl)
Phosphate buffered saline (PBS)
Erythrocyte lysis buffer (155 mmol/L NH4CI, 0.1 mmol/L EDTA, 12 mmol/L NaHCO3, with which leukocytes are isolated)
TE ( pH 7.6)
TE pH 7.6 containing 40 μg/ml of PMSF.
2. gel
Low melting point agarose (1%)
3. centrifuges and rotors
Sorvall SS-34 or equivalent rotor head
4. Specialized equipment
Gauze
Glass homogenizer with tight-fitting pestle and mortar head, pre-cooled in ice.
Cell counting plates
LeucoPrep Cell Separation Tubes (Becton Dickinson)
Mortar and pestle, pre-cooled to -70°C
42°C and 50°C water baths
5. Cells and tissues
Cell or tissue samples
II. Methods
1. Preparation of cell or tissue samples.
(1) Cultured cells
① Wash the growing cultured cells 3 times with ice-cold PBS.
① Wash the growing culture cells with ice-cold PBS 3 times. ② Collect the organelles by scraping them with a sterile rubber scraper into a small volume of ice-cold PBS.
③ Suspend the cells in ice-cold L buffer at a concentration of about 2X107 cells/ml.
(2) Fresh tissue samples
① In a Petri dish, cut the fresh tissue into small pieces (1~2 mm3 ) with a clean scalpel. Homogenize in ice-cold PBS using a pre-cooled glass homogenizer.
② Remove connective tissue fragments by filtration through two layers of gauze.
③ Wash the suspended cells three times with ice-cold PBS, and then suspend the cells in ice-cold L buffer at a concentration of about 2X107 cells/ml.
The cell number was counted using a blood cell counter.
(3) Frozen tissue samples
① Powder the frozen tissue with a -70℃ pre-cooled mortar and mix it in ice-cold PBS.
② Remove connective tissue fragments by filtration through two layers of gauze.
(iii) Wash the suspended cells three times with ice-cold PBS, and then suspend the cells in ice-cold L buffer at a concentration of about 2X107 cells/ml.
(4) Blood leukocyte samples
① Coarsely separate leukocytes and erythrocytes by centrifugation of 5-10 ml of blood in a LeucoPrep cell separator tube.
② Add 4 times the volume of erythrocyte lysate to the yellowish leukocyte layer and mix gently by inverting 2~3 times.
② Add 4 times the volume of erythrocyte lysate to the yellowish leukocyte layer and mix gently by inverting 2-3 times. ③ Warm up in buffer for 5 min at room temperature, then centrifuge at 3000 g (equivalent to a Sorvall SS-34 head at 5000 r/min) for 5 min at room temperature.
④ Suspend the precipitate with 1 ml of PBS at room temperature.
2. Prepare a volume of 1% low-melting agarose in L buffer equal to the volume of cell preparation in step 1. Cool the melted agarose to 42°C.
3. While the agarose is cooling to 42°C, warm the cell suspension (Step 1) to the same temperature. Mix the melted agarose and the cell suspension. Stir the mixture with a closed Pasteur pipette to ensure that the cells are completely and evenly dispersed in the agarose.
4. Pipette the melted mixture into a prefabricated Plexiglas mold (50-100 μl, PHarmacia or BioRad), or pipette the mixture into an appropriate length of Tygon tubing (1/8" or 3.2 mm ID) or a 1 ml plastic syringe. Leave the mold at room temperature for 15 min, then move to 4°C for 15-30 min.
5. After the agarose has set, gently collect the gel plugs from the Plexiglas molds or gently blow out the gel plugs from the Tygon tubes or syringe tubes and place them in a petri dish. Cut the cylindrical gel plugs into 1 cm long pieces.
6. Transfer the bolus into a 3-fold volume of L buffer containing 0.1 mg/ml Proteinase K and 1% (m/V) Sarkosyl and incubate at 50°C for 3 h. Replace the used buffer with a 2-fold volume of fresh buffer and continue to incubate at 50°C for 12-16 h.
7. 50-fold volume of TE (pH 7.6) is scheduled for 3-5 changes over 3 h. Incubate the gel bolus at room temperature.
8. Remove the TE and incubate the gel for 30 min at 50℃ with two times volume of TE (pH 7.6) containing 40 μg/ml PMSF.
9. Use 50-fold volume of TE (pH 7.6) and incubate the gel plug at room temperature with 3 to 5 changes of solution within 3 hours.