Storage And Stability
Gel Extraction Kit can be stored for up to 12 months at room temperature (15-25°C) or at 4°C for storage periods longer than 12 months. Any precipitate in the buffers can be re-dissolved by incubating at 37°C before use.
Gel Extraction Kit is designed to extract and purify DNA fragments of 50bp to 40kb from standard or low-melt agarose gels in either Tris acetate (TAE) or Tris borate (TBE).This membrane-based system, which can bind up to 40μg DNA, allows recovery of isolated DNA fragments in as little as 20 minutes, depending on the number of samples processed and the protocol used. The purified DNA can be used for automated fluorescent DNA sequencing, cloning, labeling, restriction enzyme digestion or in vitro transcription/translation without further manipulation.
The dissolved gel piece is placed into a Quick Gel Extraction Column containing a silica membrane. The DNA is bound to the membrane by either centrifugation or vacuum. The membrane is then washed with Wash Buffer containing ethanol to remove impurities and the purified DNA is then eluted into a Recovery Tube using Elution Buffer (10 mM Tris-HCl, pH 8.5). The purified DNA is suitable for use in a wide variety of downstream applications.
? Prior to the initial use of the kit, dilute the Wash Buffer(PE) with ethanol (100%):
After the ethanol has been added, mark the check box on the bottle to indicate the completed step.
? Examine the Gel Solubilization Buffer (BD) for precipitates before each use. Re-dissolve any precipitate by warming the solution to 37°C and cooling to 25°C.
? Freshly prepared electrophoresis buffers should be used both for gel preparation and for gel running.
? All purification steps should be carried out at room temperature.
Experienced Users Procedure
1.Weigh a 1.5ml microcentrifuge tube for each DNA fragment to be isolated and record the weight.
2.Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.
Note. If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.
3.Add Gel Solubilization Buffer (BD) at a ratio of 10μl of solution per 10mg of agarose gel slices.
4.Incubate the gel mixture at 50-60°C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversio every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved.
Note. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is red, add 10 μl of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
? High concentration gels (>2% agarose) or large gel slices may take longer than 10 minutes to dissolve.
5.Transfer the dissolved gel mixture to the Quick Gel Extraction Columns assembly and incubate for 2 minutes at room temperature.
6.Centrifuge the Quick Gel Extraction Columns assembly in a microcentrifuge at 14,000× g (12,000rpm) for 1 min, then discard the flow-through.
7.Wash the column by adding 500 μl of Wash Buffer (PE), to the Quick Gel Extraction Columns. Centrifuge the Quick Gel Extraction Columns assembly for 1 minat 14,000× g (12,000rpm), then discard the flow-through.
Note. Wash Buffer(PE) must be previously diluted with100% ethanol.
8.Wash the column by adding 500 μl of Wash Buffer (PE) again, previously diluted with 100% ethanol, to the Quick Gel Extraction Columns. Centrifuge the Quick Gel Extraction Columns assembly for 1 min at 14,000 × g (12,000rpm), then discard the flow-through.
9.Centrifuge the empty Quick Gel Extraction Columns for an additional 3 min to completely remove residual wash buffer.
10.Note. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.
11.Empty the Collection Tube and recentrifuge the column assembly for 1 min with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.
12.Carefully transfer the Quick Gel Extraction Columns to a clean 1.5ml microcentrifuge tube. Apply 50 μl of Elution Buffer (10 mM Tris-HCl, pH 8.5) directly to the center of the column without touching the membrane with the pipette tip. Incubate at room temperature for 2 minutes. Centrifuge for 1 min at 14,000× g (12,000rpm).
Note. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 μl does not significantly reduce the DNA yield. However, elution volumes less than 10 μl are not recommended.
? If DNA fragment is >10 kb, prewarm Elution Buffer to 65°C before applying to column.
? If the elution volume is 10 μl and DNA amount is >5 μg, incubate column for 1 minute at room temperature before centrifugation.
13.Discard the Quick Gel Extraction Columns and store the microcentrifuge tube containing the eluted DNA at 4°C or –20°C.