N5 and L5 Elution Buffer Preparation for High GC Content DNA

DNA with GC contents higher than 60% (or with large regions of high GC content) may continue to bind a Nucleobond AX cartridge even under normal elution conditions. In such cases, some type of DNA denaturant must be added in order to do the job. The final buffer composition that has been shown to work for the modified N5 and L5 elution buffers is as follows: 50% formamide, 1.0 M KCl, 15% EtOH, 100 mM Tris-phosphate, pH 8.5. The following procedure will make one liter of this buffer.

  1. Begin by adding 12.11g of Tris base to 250 ml of deionized water and stir to dissolve.
  2. Add 74.55g of KCl to the bring it to the required final concentration of 1.0M. Stir to dissolve and autoclave.
  3. Allow the autoclaved buffer to cool to room temperature and add 150 ml of absolute (100%) ethanol1 and 500 ml of deionized formamide. (The formamide can be deionized by treating it with a mixed bed resin to bring the pH close to 7.)
  4. Adjust the pH to 8.5 with phosphoric acid.
    NOTE: The ethanol, formamide, and phosphoric acids available to most laboratories are DNAse/RNAse free. This can be easily checked for those used in your lab. Dilute the reagent approximately 10 to 1 in DEPC-treated water and add MgCl2 to a final concentration of 10 mM. Add 1-2 mg of RNA (or DNA), incubate at 37C for 30 minutes, and run on a 1.2% agarose gel (prepared with DEPC-treated water and RNAse-free loading and running buffers, if necessary) to determine whether the RNA (or DNA) was degraded. A control lane of RNA (or DNA) in DEPC-treated water alone will determine if any degradation resulted from the gel itself.

    (Be very careful not to "overshoot" the appropriate pH for each solution when titrating because correcting it with a basic solution will alter the salt concentration of the buffer which, in turn, could alter cartridge performance.)

  5. Add enough autoclaved, distilled water to reach a final volume of 1000 ml. Store at room temperature in a tightly closed container to prevent evaporation.

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

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    Updated 1/20/98