Purification of Plasmid DNA with the NucleoSpin System

The following protocol is designed for the purification of up to 20 µg of plasmid DNA from 1-5 ml cultures of E. coli grown in LB or TB media.

Remarks:

  • Buffer A3 contains guanidine hydrochloride. The use of gloves and goggles is recommended.

  • Add RNAse A to buffer A1, mix, and store at 4C until needed.

  • The SDS in buffer A2 will precipitate at temperatures below 20C. If this occurs, store the bottle for a few minutes at 40-50C and mix well.

  • Before using the A3 buffer, incubate it 10 minutes on ice. Do not store this buffer at 4C because the salt precipitates. Add ethanol to buffer A4 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. Lower pH leads to lower recoveries.

    1. Resuspend the pellet of bacterial cells carefully in 250 µl of buffer A1.

    2. Add 250 µl of buffer A2, mix gently, and incubate at room temperature for 5 minutes. Do not vortex in order to prevent the release of chromosomal DNA from the cell debris. Do not allow the lysis to proceed for more than 5 minutes.

    3. Add 300 µl of buffer A3, mix gently by inverting the tube 6-8 times, and incubate on ice for 5 minutes. Centrifuge the suspension at greater or equal to 12,000 x g for 10-12 minutes at 4C.

    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.

    4A. When E.coli strains like HB 101, JM or any wild type strains are used, which have high nuclease activity, wash the NucleoSpin cartridge with 0.5 ml of buffer AW and centrifuge 60 sec. at maximum speed.

    5. Place the cartridge back into the tube, add 700 µl of buffer A4 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 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. Repeating this step will lead to a 15-20% higher yield. 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 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