Summary
Recently, gel electrophoresis is utilized to isolate and purify genomic DNA. And common electrophoresis supporting matrix are agarose gels and polyacrylamide gels , in which DNA is located in the three-dimensional network structure. This post is mainly to introduce methods of recovery of DNA fragments from agarose gels and polyacrylamide gels.
General principles and requirements
There are two common principles: the one is to increase the recovery efficiency of DNA fragments, and the other is to remove pollutants in DNA samples.
Recovered DNA samples can be contaminated by the impure supporting matrix and some other careless operation. In order to meet the subsequent experimental requirements, recovered DNA samples should be purified to remove contaminants. Commonly used purification methods are organic solvent extraction and commercialized column chromatography. Recovered DNA fragments usually have to be precipitated by ethanolof high concentration in the presence of salt ions Finally, washing the precipitates with 70% ethanol to remove organic molecules and co-precipitated salt.
Recovering DNA fragments from agarose gels
There are several methods for recovering DNA fragment from agarose gels, including DEAE cellulose membrane electrophoresis, electrophoresis elution method, freezing extrusion and so on.
1.DEAE cellulose membrane electrophoresis
DEAE-cellulose is a sort of anion exchange cellulose. It can be combined with negatively charged DNA molecules. Insert DEAE cellulose membrane into the gel at which it is in front of the separated nucleic acid bands after electrophoresis. Continue the process of electrophoresis until the desired and recovered DNA fragments just translocate to the membrane. Take out the DEAE cellulose membrane, and wash away the impurities under the condition of low salt-concentration. Precipitate and harvest the purified DNA molecules in the end. The method is relatively simple and can be used to recover multiple DNA fragments at the same time, especially for recovery of 500bp~5kb DNA fragments, which are highly purified and can meet the requirements of most experiments.
Electrophoresis elution method
This method is consist of two main steps: the first one is to get DNA fragments off gels after electrophoresis which then migrates into a small volume solution. The next is to isolate and purify DNA fragments. To be specific, dialysis and non-dialysis bags electrophoretic elution are two typical ways to isolate and purify DNA fragments. However, dialysis bag electrophoretic elution method is not suitable for the simultaneous recovery of multiple different DNA fragments. It can be effecient in recovering DNA fragments from 200bp to over 50kb, especially for those of larger than 5kb.
Recovering DNA fragments from polyacrylamide gels
The standard methods for recovering DNA fragments from polyacrylamide gels are through the processes of crushed or soaked. Crush the gels which contain target DNA fragments. Soak the crushed gels into the elution buffer until DNA fragments are eluted. This method can be very suit for recovering less than 1kb single-stranded or double-stranded DNA fragments, with high purity and free of enzymes inhibitors.
There are many other kinds of methods for isolation and purification of genomic DNA. Each method includes some specific protocols.
Related product: Genomic DNA Purification