In Gel Enzyme Reactions

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.

  1. Run the DNA sample on a low melting gel of the appropriate concentration. Load sufficient DNA to provide 1-10µg of target material.
  2. 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.
  3. Weigh the gel slice, and determine the approximate volume by assuming 1mg = 1µl.
  4. Heat the slice to 65°C in a sealed tube to prevent evaporation. Allow 5-10 minutes for the band to melt.
  5. 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.
  6. Allow to digest for 1-2 hours at 37°C.
  7. The digested DNA may be analyzed on a second gel, purified from the LM agarose solution, or further processed as desired.

 

In Gel Ligation
 

In most cases, the creation of cloned constructs requires the ligation of at least one, and more often two, fragments isolated from agarose gels. Isolation of bands prior to ligation prevents the generation of spurious constructs, and lowers the background of undigested vector DNA in the experiment. It is most convenient to perform ligation reactions directly in melted bands isolated from low melt agarose gels.

  1. Excise the bands to be ligated from a low melting agarose gel of the appropriate concentration. Limit the UV exposure of the DNA to under 1 minute.
     
  2. Combine the bands in a pre-weighed tube. Weigh the tube to determine the volume of agarose (assume 1mg=1µl). Melt the agarose at 65°C for 5-10 minutes, and bring to 37°C.

    If the optimal ratio of the two fragments is known (i.e. 3:1 insert:vector for subcloning), the excised bands should be melted separately, and appropriate volumes combined to give the desired ratio.

  3. Add an equal volume of 2X T4 ligase buffer, containing 1-2 units of T4 ligase. Mix well and ligate at 15°C overnight, or room temperature for 3-4 hours.

 

In Gel PCR Amplification
 

Although the PCR reaction is highly selective for the target DNA, it is often beneficial to purify the template prior to amplification. In particular, if an amplification yields multiple bands, analysis of the individual bands requires separation on agarose, which is generally followed by re-amplification to provide sufficient material for analysis.

  1. Run template DNA on a low melting agarose or high resolving gel of appropriate concentration. Stain with Ethidium Bromide and cut out the band of interst. Limit the time of exposure of the DNA to UV light to under 1 minute.

    NB: Due to the extreme sensitivity of PCR, band contamination from adjoining lanes is highly probable. Use alternating lanes to minimize this problem.

  2. Melt the DNA band at 65°C for 5-10 minutes. Add 1ml Nuclease free water for every 1µg of template DNA. Mix well.
     
  3. 1µl of the above mixture will provide 1ng of template, which is sufficient for most amplification protocols.

 

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