Isolation of DNA from mammalian cells grown in 96-well microculture plates
Isolation of DNA from mammalian cells grown in 96-well microculture plates
This protocol, modified from the method of Ramirez-solis et al. 1992, 1993, is a simple and efficient method for extracting genomic DNA from eukaryotic cells grown in each well of a microtiter plate. Each well produces enough genomic DNA for several polymerase chain reactions (PCR) or one Southern hybridization. Optimal for preparing genomic DNA for PCR reactions. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Peitang Huang et al.
Operation method
Isolation of DNA from mammalian cells grown in 96-well microculture plates
Principle
This protocol, modified from the method of Ramirez-solis et al. 1992, 1993, is a simple and efficient method for extracting genomic DNA from eukaryotic cells grown in each well of a microtiter plate. Each well produces enough genomic DNA for several polymerase chain reactions (PCR) or one Southern hybridization. Optimal for preparing genomic DNA for PCR reactions.
Materials and Instruments
Restriction Endonuclease RNase RNase without DNase Cells Move I. Materials For more product details, please visit Aladdin Scientific website.
Cell Lysis Buffer Ethanol NaCl Ethanol Solution Phosphate Buffer Sucrose Gel Sampling Buffer TE
Pumping Equipment Multi-Channel Pipettes Warm Boxes Shaking Platforms Tupperware Containers
1. Buffers and solutions
Cell lysis buffer (10 mmol/L Tris-Cl (pH 7.5), 10 mmol/L NaCl, 0.5% (m/V) Sarkosyl)
Ethanol
NaCl/ethanol solution
Phosphate buffer solution (PBS )
Sucrose Gel Sampling Buffer
TE (pH 8.0)
2. Enzyme and buffer
restriction endonuclease
RNase without DNase
3. Specialized equipment
Pumping equipment connected to a vacuum hose with valve
Multi-channel pipettes, 8 or 12 channels
Warming chamber, preset to 60°C
Shaking platforms
Tupperware container
4. Cells and tissues
Cells grown in 96-well microculture plates
II. Methods
1. Pipette the culture solution from each well of the 96-well plate with a blue-tipped or Pasteur pipette.
As long as you are careful not to touch the cell layer, it is usually not necessary to replace the tip with a new one for each well.
2. Rinse the cells in each well twice with 100 μl of phosphate buffer.
3. Using a multichannel pipette, add 50 μl of cell lysate to each well of the microplate. Place several sheets of wet blotting paper in a polypropylene box (e.g., Tupperware box), place the microtiter plate containing the cell lysate and cells on top of the blotting paper, and seal the box tightly with the lid.
4. Place the sealed box in a 60℃ incubator for 12-16 hours.
5. Remove the box from the incubator, take out the culture plate and place it flat on the bench, cool it down for a few minutes and add 100 μl NaCl/ethanol solution to each well. Without mixing, incubate the plate at room temperature for 30 min, and at the end of the incubation period, a fibrous nucleic acid precipitate should be seen.
6. Slowly invert the plate and pour the ethanol solution into the sink. The nucleic acid precipitate should adhere to the bottom of the culture wells. Invert each culture plate onto dry absorbent paper to allow residual ethanol to drain from the plate.
7. Add 150 μl of 70% ethanol to each well, being careful not to move the nucleic acid precipitate. Invert the plates as in step 6 to remove the 70% ethanol. Remove excess liquid by blotting on absorbent paper. Rinse the precipitate twice more with 70% ethanol.
8. Allow the plate to dry at room temperature until the last of the ethanol has evaporated. If the genomic DNA will be used for PCR analysis, proceed to step 9. If the DNA will be used for Southern hybridization, proceed to steps 10, 11 and 12.
9. Add 30-50 μl TE (pH 8.0) to each well and shake gently at room temperature for 12-16 h to dissolve the DNA.
10. If the DNA will be used for Southern hybridization, prepare the following restriction endonuclease mixture; 40 μl per well.
H2O 0.8 times volume
10X Restriction Endonuclease Buffer 0.1x volume
RNase without DNase 10 μg/ml
Add 10 units of restriction endonuclease per 40 μl of mixture before use.
11. Add 40 μl of restriction endonuclease reaction mixture to each well using a multichannel pipette. Repeat pipetting several times to mix the contents of the wells, being careful to avoid air bubbles. Make a wet box in a Tupperware container as in step 3, place the plate in it, and allow the reaction to incubate for 12-16 h at the appropriate digestion temperature.
12. Terminate the reaction by adding 5-10 μl of Sucrose Gel Sampling Buffer and perform Southern blotting and hybridization to analyze the digested DNA. 
