Tech Tips: GELase™ Agarose Gel–Digesting Preparation, EPICENTRE® Biotechnologies

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|Tech Tips: GELase™ Agarose Gel–Digesting Preparation|

GELase is a unique enzyme preparation developed by EPICENTRE Technologies for the recovery of intact DNA and RNA from low-melting-point (LMP) agarose gels. Yields of nucleic acids purified using GELase consistently approach 100%. The gentleness of the GELase method permits recovery of intact nucleic acids ranging in size from 50 bp to megabase chromosomal DNA. GELase-purified nucleic acids are ready for use in a variety of molecular biological applications including restriction mapping, cloning, sequencing, labeling, amplification, and transformation.

The GELase literature has two protocols - a "High Activity Protocol" and a "Fast Protocol". How do I decide which one to use?

The activity of GELase varies in different buffers. The highest activity is obtained in 1X GELase Buffer: In that buffer, 1 U of GELase digests 600 mg of a 1% molten gel in 1 hr at 45°C. In the High Activity Protocol, the gel slice is soaked in GELase Buffer for 1 hr, essentially replacing the electrophoresis buffer and giving maximal enzyme activity. We especially recommend the High Activity Protocol if you use an electrophoresis buffer such as TBE or phosphate buffer, or if you include a denaturant such as formaldehyde or glyoxal in the gel; this protocol will give the same high activity of GELase regardless of which electrophoresis buffer you used.

The "Fast Protocol" is generally recommended when you want to save time. Concentrated GELase Buffer is added directly to the gel slice, omitting the buffer exchange step of the High Activity Protocol. In this protocol, 1 U will digest 300 mg of molten 1% gel in 1 hr at 45°C for gels made with TAE buffer, but the total time for nucleic acid recovery is less than that of the High Activity Protocol. (The Fast Protocol can be made even faster. See below.)

What factors are most important to assure complete digestion of an agarose gel slice?

First, be sure to use the right amount of GELase according to the gel buffer and the protocol you choose. Beyond that, the most critical factor for complete digestion of a gel slice is to be sure that all of the gel is completely melted prior to adding GELase and that it remains molten throughout the digestion.

During the process of gelling, the agarose polysaccharides wrap around each other to form helices and then these helices wrap around each other to form bundles of helices. Thus, in its gelled state, many of the chemical bonds of agarose which would be cleaved by GELase are buried inside these helical bundles and are inaccessible to the enzyme. This is why it is so important to obtain complete melting before beginning the digestion and to prevent re-gelling during it.

How can I assure that the gel is completely melted?

First, the time needed for melting at 70°C depends on the tube or container used. In general, glass conducts heat much better than plastic, and tubes with thinner walls take less time to melt than with thicker walls. Next, be sure to immerse the tube in the water bath or heating block so the walls are heated well above the gel surface. Avoid any action which will splash molten agarose onto the tube cap or unheated tube walls such as vortexing. Breaking up a gel slice >300 mg into pieces will shorten the melting time. If you use the Fast Protocol, add the GELase Buffer before melting since cold buffer added later could cause localized re-gelling. Longer melting times are required for agarose concentrations greater than 1%. Once melting at 70°C is complete, transfer the tube to a 45°C water bath for equilibration prior to adding GELase. (Allow adequate time for equilibration. GELase will be inactivated if exposed to temperatures higher than 45°C). To avoid re-gelling, don't remove the tube from the water bath for more than a few seconds to add the GELase or perform other manipulations. Even better, add the enzyme and mix by gentle stirring with the pipet tip with the tube still in the 45°C bath.

What can I do to assure maximal yields with GELase?

In most cases, yields from the GELase procedure approach 100%. The nucleic acids stay in the original tube throughout the digestion – no transfer to columns or filters where losses can occur. However, there are two important points to consider in obtaining the highest possible yields. First, the DNA pellets are often transparent and only loosely attached to the tube after ammonium acetate/ ethanol precipitation. Carefully remove the supernatant with a pipette rather than by decanting. Second, fresh ammonium acetate is important for the precipitation; make solutions from the pure salt which has been kept cool in a closed container. Store the filter-sterilized solution at +4°C.

More information is available on the recovery of very small or very large DNAs or recovery from dilute solutions (<0.5 µg/ml) by contacting Technical Service.

Can I use salts or alcohols other than ammonium acetate and ethanol to precipitate my DNA after GELase extraction?

Yes, however after evaluating many combinations of salts and alcohols for precipitation of nucleic acids after a GELase reaction, we find that ammonium acetate and ethanol are superior in avoiding coprecipitation of the small oligosaccharides resulting from GELase digestion of the agarose . Using other salts or alcohols can precipitate these oligosaccharides and contaminate the DNA or RNA. However, if the purified DNA or RNA is to be subsequently 5´-end labeled using T4 Polynucleotide Kinase, it is especially important to remove all residual ammonium ions, as ammonium salts are strong inhibitors of the enzyme. In this case, either perform the precipitation step using only ethanol after the GELase digestion, or, if ammonium acetate is used, wash the RNA or DNA pellet once with 70% ethanol before the kinase step.

GELase™: How to Do the Fast Protocol Even Faster

The GELase Fast Protocol was developed to speed recovery of nucleic acids from low-melting-point agarose gels and deliver quantitative yields. The general protocol will handle gels made in a variety of buffers, a range of agarose concentrations, gel slices of any size, and DNA or RNA of all molecular weights using methods proven to be successful for a wide range of conditions. On narrowing the scope of conditions covered by the Fast Protocol, the time required for some steps can be reduced to significantly decrease recovery time of nucleic acids. The standard Fast Protocol is described in Figure 1 and is used as the reference point for modifications. Remember, the general protocol has "margins of safety" built into it to assure nearly 100% yield. EPICENTRE recommends that modifications be tested after first using the standard Fast Protocol.

The standard Fast protocol can be used to digest gels made in TAE, TBE, and phosphate buffer. However, the activity of the enzyme is reduced by some components in these buffers (under standard conditions, 1 U of GELase will digest 300 mg of 1% LMP-agarose made with TAE or 80 mg made with TBE or 120 mg made with phosphate buffer). To optimize enzyme activity for this protocol, we used gels made in TAE .

Gel slices can be melted in plastic tubes when sufficient time is allowed for the slower conductance of heat through plastic. The Fast Protocol recommends 20 min to obtain complete melting of the gel slice which is essential for complete digestion. To reduce melting times, use glass tubes and gently break the gel slice into several pieces with a clean spatula. Table 1 lists melting times for several sizes of 1% gel when heated in glass. Please note, longer melting times are required for gels with higher agarose concentrations.

Table 1. Melting times of 1% LMP–agarose in glass tubes
Gel Slice	Melting Time
300 mg	3 min
500 mg	5 min
1000 mg	7 min

GELase enzyme is inactivated by temperatures higher than 45°C and cannot completely digest agarose that has begun to regel. For these reasons, the entire gel sample must be cooled to 45°C in a water bath before adding enzyme. Ten minutes is the suggested equilibration time for most sample sizes in the general protocol. More precise cooling times for several sample sizes is given in Table 2. These times apply to 1% agarose melted in glass containers.

Table 2. Equilibration times of 1% LMP–agarose in glass tubes
Gel Slice	Equilibration Time
300 mg	2 min
500 mg	2 min
1000 mg	3 min
5000 mg	6 min

The standard protocol is based on 1U of enzyme digesting a quantity of gel, taking into account the percent of agarose and the electrophoresis buffer used. Gel digestion will occur more quickly when more enzyme is used. Table 3 shows the relative digestion times for a 300 mg gel slice under increasing amounts of enzyme. For larger gel slices or faster digestion, more enzyme can be added. Also, when adding more enzyme be sure the enzyme is thoroughly mixed into the solution.

Table 3. Digestion time for a 300 mg gel slice of 1% agarose
Desired Digestion Time	Units GELase
60 min	1
30 min	2
15 min	3

Our standard protocol uses a centrifugation time of 30 min to achieve high yield. When precipitating RNA or DNA over 500 bp in size and above 0.5 µg/ml in concentration during ethanol precipitation, good recovery can be achieved with shorter centrifugation times. However, nucleic acids less than 500 bp or in dilute solution, will require 30 min for quantitative recovery. Table 4 lists reduced centrifuge times for DNA or RNA recovery in several size ranges.

Table 4. Reduced centrifugation times after GELase Digestion

Size of DNA or RNA Minimum Time

One kilobase or greater 5 min

500 bp–1 kbp 15 min

< 500 bp 30 min