The extraction of DNA from agarose gel has many downstream applications in molecular biology research work and biomedical research. For instance, we often need to sequence or clone the DNA fragments obtained either by PCR or restriction digestion. Therefore, it is necessary to have a pure DNA sample to have better sequencing or for better transformation efficiency.
Though there are many protocols for DNA separation such as DNA purification using electrophoresis and column chromatography. Among these, DNA separation by gel electrophoresis is the most common and easier one to perform with many advantages. So, here I’m going to mention some of the methods involving DNA separation by gel electrophoresis.
DNA separation by gel electrophoresis normally involves the use of agarose gel. It’s simple, convenient, and easy to purify DNA fragments. For the extraction of DNA from agarose gel, a low melting point agarose is preferable. The concentration of the agarose gel for the gel extraction procedure ranges from 0.7 to 0.8 %.
Low melting point agarose melts at a lower temperature as compared to standard agarose. Therefore, it preserves the integrity of the double stranded DNA as high temperature might denature the double-strands. Moreover, a lower concentration of agarose provides much easier electrophoretic separation of the DNA. It is also easier to dissolve the gel in the extraction buffer for purification purposes.
Gel extraction of DNA can also be done in many ways. Such as, 1) Gel excision followed by purification and concentration, 2) In-gel isolation of the DNA fragments that don’t require purification and concentration (ready-to-use).
Purification of DNA involving gel excision
Gel excision of DNA involves the electrophoretic fractionation of the DNA. After electrophoresis, excise the band of desired DNA from the gel under a UV transilluminator. The piece of the gel containing the desired DNA fragment will be used for DNA extraction in the next step. The gel excision process involves the dissolution of the gel piece into an appropriate binding buffer followed by purification.
During this process, the agarose gel is removed leaving behind the desired DNA in an elution buffer. Thus eluted DNA samples can directly be used for downstream applications. DNA from the bands excised from the agarose gel can be purified either of two ways; using commercially available gel extraction kits or conventional methods of gel extraction.
Purification of DNA involving In-gel isolation
This is the most advanced method for the extraction of DNA from agarose gel. In this method, the desired DNA fragment can be isolated directly from the gel while doing the electrophoresis. To do so, electrophoresis is integrated with a built-in transilluminator such as a MupidTM-One LED illuminator. These instruments provide real-time monitoring of DNA migration.
These setups are designed in such a way that provides real-time visualization of DNA migration. These are highly effective to isolate the DNA directly from the gel with no further purification needed. To do so, prepare the 1 % agarose gel of standard concentration and make two wells system; sample well and collection well. The collection wells lie at a certain distance below the corresponding sample well. The collection wells are always near the positive (+) terminal. Each collection well aligns with its respective sample wells.
During the electrophoresis, load the DNA sample into the sample well and fill up the collection well with TE buffer. Fill up all other blank wells with distilled water to avoid the electrophoresis error. After an appropriate time, the desired DNA fragment reaches the collection well from where the DNA sample can be collected using an adjustable micropipette.
Well, this is also a type of electroelution but it greatly differs from the conventional electroelution methods. This is because you don’t need to excise the DNA band from the gel but you can directly isolate the DNA from the agarose gel. This method is quite expensive but it gives fast track of DNA isolation that is because it eliminates the gel excision and purification steps. Since the DNA comes out in the TE buffer, it is ready-to-use that can be used in downstream applications. Watch this YouTube tutorial to understand how in-gel DNA isolation works.