Direct Purification of PCR Products 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 recovery rate is pH dependent. The pH of the elution buffer must be 8.5 in order to obtain maximum yields. A lower pH leads to lower recoveries.

    1. The removal of mineral oil is not necessary. (It can be performed fairly easily in a number of ways without chloroform extraction. For example, the PCR sample can be incubated at -20C for about 30 minutes allowing the aqueous phase to freeze. The mineral oil can then be pipetted off.)

    2. Adjust the volume of the reaction mixture to 100 µl using TE, pH 8.0. Add 400 µl of buffer NT2 to the sample and mix.

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

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

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

    6. Place the NucleoSpin cartridge into a clean micro centrifuge tube (1.5 or 2.0 ml). Add 50 µl 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.

    6a. 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 µl through the warmed assembly.

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

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    Updated 1/20/98