In many cases, the processing of DNA by enzymes is not impeded by agarose. Such reactions can be run directly in bands excised from
low melting point agarose gels. The excised band is melted, mixed with the required buffer and enzyme, and then incubated at the optimal reaction temperature. The gel may solidify during the incubation without interfering with the reaction, and the agarose can then be remelted to recover the DNA. Restriction digests, digestions and PCR amplifications can all be run as in-gel reactions.
NB:
TAE,
TBE and
TTE, as well as most other common electrophoresis buffers, contain EDTA at 1-2mM. This level of EDTA is sufficient to inhibit most DNA modifying enzymes, by chelating the Mg
++ that these enzymes require. The effect of the EDTA can be avoided in two ways: more Mg
++ may be added to the reaction to saturate the EDTA, or less EDTA may be included in the electrophoresis buffer. Impure samples of DNA may be degraded by endogenous nucleases if the EDTA is decreased in the gel buffer, so if the purity of the sample is questionable, it is advisable to run the gel in the standard buffer and add supplementary Mg
++ to the subsequent reaction.
In Gel Restriction Digestion
This technique is used when one DNA species from a complex mixture is to be digested. Without pre-purification, digestion would result in a complex mass of unidentifiable bands.
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Run the DNA sample on a low melting gel of the appropriate concentration. Load sufficient DNA to provide 1-10µg of target material.
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Stain the gel with Ethidium Bromide, and cut out the band(s) of interest. Do not expose the DNA to UV light for more than 1-2 minutes, or nicking and strand breaks may occur.
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Weigh the gel slice, and determine the approximate volume by assuming 1mg = 1µl.
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Heat the slice to 65°C in a sealed tube to prevent evaporation. Allow 5-10 minutes for the band to melt.
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Equilibrate the melted band to 37°C, and add an equal volume of 2X enzyme buffer containing 5-10 units of enzyme per µg DNA in the slice.
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Allow to digest for 1-2 hours at 37°C.
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The digested DNA may be analyzed on a second gel, purified from the LM agarose solution, or further processed as desired.
