Restriction endonuclease digestion of agarose-embedded DNA has proven to be a valuable tool in the physical mapping of chromosomes. To compare restriction endonuclease performance under controlled conditions, purified substrate DNAs (lambda, adenovirus-2) were embedded in 1% ImBed® low-melting-point agarose and subsequently digested. The results are presented below.
Substrate DNAs were embedded in 1% ImBed agarose (1 µg DNA/30 µl). The GelSyringe™ agarose-
dispensing system was used to produce 30 µl agarose plugs. The agarose plugs were equilibrated in the recommended 1X NEBuffer by two 15 minute washes (100 µl each) and then incubated in 100 µl of fresh 1X NEBuffer plus 2, 5, or 20 units of enzyme for 2, 4 or 16 hours (total reaction volume of 130 µl). Most enzyme reactions were incubated at 37°C; thermophilic enzymes (D) were incubated at 50°C. After digestion, cold TE [10 mM Tris-HCl, 0.5 mM EDTA] was used to stop the reactions. The resulting digests were resolved on 1% agarose gels and compared to a standard digest (1 µg, 50 µl liquid reaction volume, 1 hour).
The values reported are the minimum units required for complete digestion. An asterisk (*) indicates that digestion was incomplete at that time point with the maximum number of units used in these tests (20 units). In some cases, the experiment was repeated after a 16 hour preincubation (at 4°C) to help diffuse the enzyme into the plug. Values in parentheses indicate the minimum units required for complete digestion after preincubation. It should also be noted that the actual number of units required for complete digestion will vary depending on the DNA substrate and its level of purity.
Limited enzyme diffusion into the agarose matrix may account for diminished enzyme performance on agarose-embedded DNA. To reduce this effect, plugs should be made with low-melting-point agarose at concentrations of 0.5-1.0%. A 16 hour preincubation of the reaction (at 4°C) is useful in cases where enzyme stability is compromised by incubation at 37°C for extended periods of time. Gentle mixing of the reaction has also been reported to aid in the diffusion of enzyme into the plug.