Restriction endonuclease digestion of DNA in agarose gel plugs
Chromosomal DNA isolated from mammalian cells, yeast or bacteria can be digested in agarose plugs with the desired endonuclease. The low melting point agarose plugs are sufficiently porous to allow the endonuclease to enter and interact with the genomic DNA as a substrate. After digestion, the DNA is separated by PFGE and recovered or analyzed by Southern blotting. This experiment is from "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Restriction endonuclease digestion of DNA in agarose gel plugs
Principle
Chromosomal DNA isolated from mammalian cells, yeast or bacteria can be digested in agarose plugs with the desired endonuclease. The low melting point agarose plugs are sufficiently porous to allow the endonuclease to enter and interact with the genomic DNA as a substrate. After digestion, the DNA is separated by PFGE and then recovered or analyzed by Southern blotting.
Materials and Instruments
Restriction Enzyme Genomic DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Restriction enzyme buffer TE
Gel for pulsed-field electrophoresis Water bath
1. Buffers and solutions
1X restriction enzyme buffer
TE ( pH 7.6)
2. Enzyme and buffer
Restriction enzymes
3. gels
Gel for pulsed field electrophoresis
4. Nucleic acids and oligonucleotides
Genomic DNA embedded in low melting point agarose plugs
5. Specialized equipment
Water bath set at the optimal temperature for enzyme digestion
II. Methods
1. If the gel plugs have not been stored in TE (e.g., received in the mail or have been stored in 0.5 mol/L EDTA), incubate the gel plugs in a 50x volume of TE (pH 7.6) at room temperature for 30 min. transfer the gel plugs to a new 50x volume of TE (pH 7.6) and incubate the gel plugs at room temperature for a second 30 min. otherwise, proceed directly to step 2.
2. Transfer gel plugs to microcentrifuge tubes and add 10x volume of the appropriate 1X Restriction Buffer to each tube. Incubate at room temperature for 30 min.
3. Remove the buffer and replace it with 2 to 3 times the volume of fresh 1X Restriction Buffer. Add 20~30 units of the corresponding restriction enzyme per tube and incubate at the optimal temperature for the enzyme's action; in the case of YAC DNA, incubate for 3 h, and in the case of mammalian DNA, incubate for 5~6 h.
4. If the DNA is digested with only one enzyme, after digestion, immerse the gel plug in 20 times the volume of TE (pH 7.6) at 4°C for 1 h and then follow step 7. If the DNA is digested with more than one enzyme, skip the TE incubation and proceed to step 5.
5. If the DNA is digested with more than one enzyme, equilibrate the gel bolus again with the appropriate buffer before adding the second enzyme. To fully equilibrate, use an automated pipette to remove as much of the first enzyme buffer as possible from each tube and replace it with 1 ml of TE. Remove the TE and add fresh 1 ml TE. Store at room temperature for 30-60 min. gently remove the TE.
6. Add 10 times the volume of the second 1x Restriction Enzyme Buffer to each tube and incubate at room temperature for 30 min. perform restriction enzyme digestion as described in step 3. Finally, each gel plug is immersed in 20x volume of TE (pH 7.6) at 4°C for 1 h. The tubes are then incubated for 30 min at room temperature with 10x volume of the second 1x restriction enzyme buffer.
7. Using a disposable pipette tip, gently push the gel plugs directly into the spiking wells (slots) of the pulsed-field gel for electrophoretic separation of DNA fragments.