Removal of small fragments of nucleic acids from plasmid DNA preparations by NaCl centrifugation
Removal of small fragments of nucleic acids from plasmid DNA preparations by NaCl centrifugation
Conventional plasmid DNA preparations are contaminated, to varying degrees, with small molecules of DNA or RNA from the genome of the host bacterium or plasmid breakage fragments, which are small in molecular mass but large in number, and can significantly affect the total number of DNA fragments at the 3' and 5' ends of the preparations. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Removal of small fragments of nucleic acid from plasmid DNA preparations by NaCl centrifugation
Principle
Conventional plasmid DNA preparations are contaminated, to varying degrees, with small molecules of DNA or RNA from the genome of the host bacterium or from plasmid breakage fragments, which, although not large in molecular mass, are numerous enough to significantly affect the total number of DNA fragments at the 3' and 5' ends of the preparation.
Materials and Instruments
Ethanol NaCl Sodium acetate TE Tryptic RNase Move I. Materials For more product details, please visit Aladdin Scientific website.
Beckman SW 50.1 rotor
1. Buffers and solvents
Ethanol, NaCl (1 mol/L, prepared with TE ( pH 8.0)), sodium acetate (3 mol/L, pH 5.2), TE ( pH 8.0).
2. Enzyme and buffer
Tryptic RNase without DNase
3. nucleic acids and oligonucleotides
DNA samples, purified by density gradient centrifugation with CsCl
4. centrifuge and rotor
Beckman SW 50.1 rotor or alternative rotor that can be adapted to the appropriate tube, Sorvsll SS-34 rotor or alternative rotor with the same performance.
1. Determine the volume of the plasmid preparation. Add 0.1x volume of 3 moI/L sodium acetate (pH 5.2) and 2x volume of ethanol, mix well, and allow to stand at 4°C for 30 min.
2. Centrifuge at >10,000 g (with a Sorvall SS-34 rotor at >9100 r/min) for 15 min at 4°C to recover the nucleic acid precipitate, remove as much of the supernatant as possible, and leave the tube open on a bench for a few minutes to evaporate the last trace of ethanol remaining.
3. Dissolve the moist DNA precipitate with 0.5-1 ml TE (pH 8.0).
The concentration of plasmid DNA should be not less than 100 μg/ml.
4. Add RNase without DNase to a final concentration of 10 μg/ml and incubate for 1 h at room temperature.
5. Prepare a Beckman SW 50.1 centrifuge tube (or equivalent) by adding 4 ml of TE (pH 8.0) containing 1 mol/L NaCl, and using an automated pipette with a disposable tip to spread a layer of 1 ml of RNAase-treated plasmid DNA on top of the 1 mol/L NaCl solution. If necessary, fill the centrifuge tube with TE (pH 8.0) as appropriate.
6. Centrifuge at 20°C for 6 h at >150,000 g (with a Beckman SW50.1 rotor at >40,000 r/min) and carefully remove the supernatant.
The plasmid DNA settles to the bottom of the tube, while the oligonucleotides and small fragments of DNA remain in the supernatant.
7. Re-dissolve the plasmid DNA precipitate with 0.5 ml TE (pH 8.0), add 50 μl of 3 mol/L sodium acetate (pH 5.2), and transfer the DNA solution to a microcentrifuge tube.
8. Add 2 times the volume of ethanol and leave at 4°C for 10 min to precipitate the DNA, then centrifuge at >10,000 g for 15 min at 4°C to remove as much of the supernatant as possible, open the tubes, and leave them on a bench for a few minutes to allow the last traces of ethanol to evaporate.
9. Dissolve the moist plasmid DNA precipitate with TE (pH 8.0).