Sephacryl S-1000 Chromatography for Removal of Small Fragments of Nucleic Acids from Plasmid DNA Preparations
Sephacryl S-1000 Chromatography for Removal of Small Fragments of Nucleic Acids from Plasmid DNA Preparations
Sephacryl S-1000 chromatography is an alternative method for separating plasmid DNA from small molecules of nucleic acids (DNA and RNA). This method was first used by F. DeNoto and H. Goodman at Massachusetts General Hospital, Boston, and later summarized in a paper by Gomez-Marquez et al. in 1987. This experiment is from "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Sephacryl S-1000 Chromatography for Removal of Small Fragments of Nucleic Acids from Plasmid DNA Preparations
Principle
Sephacryl S-1000 chromatography is an alternative method for separating plasmid DNA from small molecules of nucleic acids (DNA and RNA). This method was first used by F. DeNoto and H. Goodman at Massachusetts General Hospital, Boston, and later summarized in a paper by Gomez-Marquez et al. in 1987.
Materials and Instruments
DNA Sample Move I. Materials For more product details, please visit Aladdin Scientific website.
Bromophenol Blue Sucrose Solution Ethanol Phenol Sephacryl Equilibration Buffer Sodium Acetate TE Agarose
Sorvall SS-34 rotor or other rotor of equal performance
1. Buffers and solvents
Bromophenol blue sucrose solution, ethanol, phenol, Sephacryl equilibration buffer, sodium acetate (3 mol/L, pH 5.2), and TE (pH 8.0) containing RNase A at a concentration of 20 μg/ml.
2. Gel
0.7% agarose, prepared with TBE.
3. nucleic acids and oligonucleotides
DNA samples were purified by density gradient centrifugation with CsCl
4. centrifuge and rotor
Sorvall SS-34 rotor or other rotor of equal performance.
5. Specialized equipment
Chromatography column filled with Sephacryl resin, Sephacryl S-1000 gel filtration resin (Pharmacia)
Methods
1. Prepare a 1x10 cm Sephacryl S-1000 column, equilibrated with Sephacryl Equilibration Buffer.
2. Determine the volume of plasmid preparation. Add 0.1 times the volume of 3 mol/L sodium acetate (pH 5.2) and 2 times the volume of ethanol, mix well, and place at 4℃ for 30 min.
3. Recover the nucleic acids by centrifugation at >10,000 g (equivalent to 9100 r/min with a Sorvall ss-34 turntable) for 15 min at 4°C. Remove as much of the supernatant as possible, open the tubes, and let them stand for a few minutes on the bench to evaporate the last traces of ethanol.
4. Re-dissolve the moist plasmid DNA precipitate with a small amount of TE (pH 8.0) (up to 400 μl) containing RNAase I. The final concentration of RNAase should be at least 100 μg/ml.
5. Incubate the plasmid DNA solution for 1 h at room temperature.
6. Extract once with an equal volume of TE (pH 8.0) equilibrated with phenol.
7. Aspirate the upper aqueous solution and add 100 μl of Bromophenol Blue Sucrose Solution. Spread the blue layer onto a Sephacryl S-1000 column.
8. Load the plasmid DNA solution onto the column and wash the column with Sephacryl Equilibration Buffer to immediately begin the fractional collection of the 0.5 ml component.
9. After 15 fractions have been collected, clamp the bottom of the column so that the blue dye flows through exactly half of the column.
10. Take a 10 μl sample from each of the collected fractions and electrophoresis with 0.7% agarose and fluorescent staining with ethidium bromide to determine the fraction containing plasmid DNA.
11. Combine the fractions containing plasmid DNA, add 2x the volume of ethanol, and precipitate for 10 min at 4°C. The DNA precipitate is then recovered by centrifugation at >10,000 g for 15 min at 4°C (equivalent to a Sorvall SS-34 turntable at 9200 r/min).
12. Remove as much of the supernatant as possible, open the tubes, and leave them on the bench for a few minutes to evaporate the last remaining traces of ethanol.
13. Dissolve the moist plasmid DNA precipitate with TE (pH 8.0).