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 Protocol for the Recovery of DNA from Low Melting Point Agarose Gels
(with Agarase)

1. Perform electrophoresis of DNA in a low melting point (LM) agarose gel prepared in TAE, 0.5X TBE, TBE or TPE buffer. Stain the gel with ethidium bromide.
2. Under UV light, cut out the desired band from the agarose gel with a clean, nuclease free scalpel*. Cut out only as much agarose as is necessary to recover DNA band.

   Note  
   For subcloning of gel-purified DNA fragments care should be taken to avoid DNA damage with UV light. Always use long wavelength (360 nm) UV light-box during preparation of agarose gel slice. If only short-wavelength (254-312 nm) UV light-box is available, minimize the UV exposure to several seconds or keep the gel on glass or plastic plate when on transilluminator.

3. Place the gel slice into a pre-weighed 1.5 ml microcentrifuge tube and determine the weight of the slice. To facilitate melting, cut gel slices larger than 200 mg into smaller pieces.
4. Incubate the tube at 70°C for approx. 10 min until the agarose is completely melted. If the agarose is not completely melted, hydrolysis will also be incomplete.
5. Transfer the tube to a 42°C water bath and equilibrate for 5 min prior to adding Agarase.
6. Add 1 unit Agarase per 100 mg (approx. 100 µl) of 1% agarose, gently mix and incubate at 42°C for 30 min. If you are using a higher percentage agarose, the amount of Agarase should be proportionally increased.
7. Add ammonium acetate** to 2.5 M concentration, chill on ice for 5 min.
8. Centrifuge at 10,000 rpm for 10 min to pellet undigested carbohydrates. Transfer the supernatant to a clean tube.
9. Add 2-3 volumes of ethanol or 0.8 volume of isopropanol, mix gently and incubate at room temperature for 1 hour. If DNA fragments are smaller than 500 bp or if DNA concentration is lower than 0.05 µg/ml, incubate at room temperature for 2 hours.
10. Centrifuge at 10,000 rpm for 15 min, remove supernatant and dry pellet. The pellet can be resuspended in TE or an appropriate buffer for subsequent manipulation.

Note  
* Alternatively, visible dyes can be included in standard agarose gels to visualize DNA bands in ambient light (2, 3).
** The T4 Polynucleotide Kinase is inhibited by ammonium ions. Use sodium acetate (0.3 M final concentration) if labeling 5’-ends of DNA with T4 Polynucleotide Kinase will be subsequently performed. In other cases, to avoid co-precipitation of oligosaccharides with DNA, it is recommended to use ammonium acetate rather than other salts.

Large DNA fragments (>30 kb) require delicate handling to avoid mechanical shearing. After Agarase digestion centrifuge at maximum speed for 10 min to pellet undigested carbohydrates. Remove oligosaccharides and Agarase by dialysis or carry out subsequent manipulations with DNA in the digested agarose solution.

References

1. Yaphe, W., The use of agarase from Pseudomonas atlantica in the identification of agar in marine algae (Rhodophyceae), Can. J. Microbiol., 3, 987-993, 1957.
2. Rand, K.N., Crystal Violet can be used to Visualize DNA Bands during Gel Electrophoresis and to Improve Cloning Efficiency, Elsevier Trends Journals Technical Tips Online, T40022, 1996.
3. Adkins, S., Burmeister,M., Visualization of DNA in agarose gels and educational demonstrations, Anal. Biochem., 240 (1), 17-23, 1996.

Ordering Information

Agarase TopVision™ LM GQ Agarose InsTAclone™ PCR Cloning Kit CloneJET™ PCR Cloning Kit T4 DNA Ligase T4 RNA Ligase Rapid DNA Ligation Kit Rapid DNA Ligation & Transformation Kit Glycogen Water, nuclease-free

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Updated sausio 16, 2008 15:32