Extraction of DNA from Agarose Gels Using the NucleoSpin Extract 2 in 1 System


Remarks:

  • The NucleoSpin Extract kit should be stored at 4C. Storage at 4C causes salt precipitation.

  • Add ethanol to buffer NT3 as specified on the tube before use.

  • The following protocol is designed for the purification from both TAE and TBE agarose gels. Normal as well as low melting agarose gels can be used

  • The recovery rate is pH dependent. The pH of the elution buffer must be 8.5 in order to obtain maximum yields. Lower pH leads to lower recoveries.


    1. Using a clean scalpel or razor blade, gently excise the DNA fragment in order to minimize excess agarose. Determine the weight of the gel slice and transfer it to a clean micro centrifuge tube.

    2. For each 100 mg of agarose gel add 300 Ál buffer NT1. For highly concentrated agarose gels, add an additional 300 Ál for each percent over 1%. (For example, for a 2% gel use 600 Ál, for a 3% gel use 900 Ál, and so on.)

    3. Incubate the sample for 10 minutes at 50C, vortexing it every 2-3 minutes.

    4. Place a NucleoSpin cartridge in a 2 ml micro centrifuge tube and load the supernatant from step 3 onto the cartridge. Centrifuge 60 seconds at maximum speed and discard the flowthrough.

    5. Place the cartridge back into the tube, add 750 Ál of buffer NT3 to the cartridge, and centrifuge 60 seconds at maximum speed.

    6. Discard the flowthrough and centrifuge the cartridge once more to remove residual ethanol from the wash buffer.

    7. Place the NucleoSpin cartridge into a fresh micro centrifuge tube (1.5 or 2.0 ml). Add 50 Ál of TE, pH 8.5 or 10mM Tris-HCl, pH 8.5 to the cartridge and centrifuge for 60 seconds at maximum speed to elute the DNA. (This step may be repeated to improve the yield, if necessary.) The DNA is ready to use.

    7a. For increased recoveries of high GC content DNA or larger DNA (5-50 kb), add Tris pH 8.5 to the cartridge/centrifuge tube assembly and incubate in a water bath for 10 minutes at 55C. Vortex or swirl it for a few seconds prior to centrifuging the 50 Ál through the warmed assembly.

    Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.

    Back to the Nucleobond AX applications guide


    Updated 1/20/98