Purification of DNA recovered from agarose and polyacrylamide gels by anion-exchange chromatography
Purification of DNA recovered from agarose and polyacrylamide gels by anion-exchange chromatography
DNA fragments recovered from agarose gels, such as PCR products or digested DNA fragments, are resistant to further digestion. The reason for this resistance is multifactorial and is usually attributed to the presence of inhibitors. Purification of double-stranded DNA by positively charged ion exchange resins effectively removes inhibitors from the sample. In a low ionic strength buffer, the negatively charged DNA binds to the positively charged substrate. The source of this experiment is "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Purification of DNA recovered from agarose and polyacrylamide gels by anion-exchange chromatography
Principle
DNA fragments recovered from agarose gels, such as PCR products or digested DNA fragments, are resistant to further digestion. The reason for this resistance is multifactorial and is usually attributed to the presence of inhibitors. Purification of double-stranded DNA by positively charged ion exchange resins effectively removes inhibitors from the sample. In a low ionic strength buffer, negatively charged DNA binds to a positively charged substrate, such as DEAE-Sephacel or DEAE-Sephadex, and the contaminant is eluted, and then the DNA is eluted from the substrate by increasing the ionic strength of the buffer. Since plasmid DNA and single-stranded DNA are difficult to elute when bound to a substrate, purification with DEAE resin is not recommended.
Materials and Instruments
DNA Sample Move I. Materials For more product details, please visit Aladdin Scientific website.
Ethanol Isopropanol Phenol Chloroform TE
Disposable cartridges or 2 ml syringes Resin, pre-swollen DEAE-cellulose or DEAE-Sephacel
1. Buffers and solutions
Ethanol
Isopropyl alcohol
Phenol: chloroform (1:1, V/V)
TE ( pH 7.6)
TE ( pH 7.6) containing 0.1 mol/L NaCl
TE (pH 7.6) containing 0.2 mol/L NaCl
TE (pH 7.6) containing 0.3 mol/L NaCl
TE (pH 7.6) containing 0.6 mol/L NaCl
2. Nucleic acids and oligonucleotides
DNA samples should be dissolved in TE (pH 7.6).
3. Specialized equipment
Disposable cartridges or 2 ml syringes
Resin, pre-swollen DEAE-cellulose or DEAE-Sephacel
II. Methods
1. Suspend DEAE in 20 times the volume of TE (pH 7.6) containing 0.6 mol/L NaCl. After the resin settled, the supernatant was aspirated. The resin was gently resuspended by adding another 20 times the volume of TE (pH 7.6) containing 0.6 mol/L NaCl. After the resin resettles, aspirate most of the supernatant. The equilibrated resin can be stored at 4℃.
2. Take 0.6 ml (enough to bind 20 μg of DNA) of DEAE suspension and fill a small column or 2 ml syringe barrel.
3. Wash the column separately with the following solutions:
3 ml of TE (pH 7.6) in 0.6 mol/L NaCl, 3 ml of TE (pH 7.6), 3 ml of TE (pH 7.6) in 0.1 mol/L NaCl, 3 ml of TE (pH 7.6).
4. Mix DNA (dissolved in TE (pH 7.6)) with an equal volume of TE (pH 7.6) containing 0.2 mol/L NaCl, sample the mixture onto a chromatography column, collect the effluent and reload the column.
5. 1.5 ml of TE (pH 7.6) containing 0.3 mol/L NaCl was washed twice.
6. 0.5 ml of TE (pH 7.6) containing 0.6 mol/L NaCl was used to elute the DNA for 3 times.
7. Extract the eluate with phenol/chloroform once.
8. Dispense an equal volume of water into two microcentrifuge tubes, add an equal volume of isopropanol to each tube, leave at room temperature for 15 min, and then centrifuge at 4℃ for 10 min at maximum speed to recover DNA.
9. Wash the precipitate carefully with 70% ethanol, open the mouth of the centrifuge tubes, and place them on a bench for a few minutes to allow the ethanol to evaporate. The DNA was then redissolved in a small volume (3~5 μl) of TE (pH 7.6).
10. Characterize and quantify the DNA fragments by polyacrylamide gel and agarose gel electrophoresis.
(1) Mix a small amount (estimated to contain 20 ng) of finalized DNA fragments with 10 μl of TE (pH 8.0) and add 2 μl of the desired gel loading buffer.
(2) Add samples and perform electrophoresis on polyacrylamide and agarose gels at appropriate concentrations, using a known amount of the original DNA digested with an appropriate restriction enzyme as a standard reference. Isolated DNA fragments should migrate synchronously with the correct fragments in the restriction enzyme digest.
(3) Carefully examine the gel for the presence of a weak fluorescent band. The presence of a weak fluorescent band indicates DNA contamination. The amount of DNA in the final product can often be estimated from the relative fluorescence intensity of the isolated fragments to a standard reference.
In rare cases, the recovered DNA will need to be further purified. This is best accomplished by reusing DEAE-Sephacel chromatography or by using specific resins from different manufacturers. Many of the resins are pre-formed into small columns suitable for microcentrifugation to purify small amounts of DNA, and are more suitable for purifying linear DNA molecules than cyclic plasmid DNA.