These columns contain silica matrices or membranes that can bind nucleic acids in the presence of high salt. Further, the silica matrix is often chemically modified by attaching strong anionic groups such as the diethylaminoethyl (DEAE) to enhance the column binding capacity for nucleic acids. Although the columns are expected to be discarded after a single use, considering the costs involved, a safe and simple strategy is required to permit their reuse. The most serious concern about reusing the columns is the imminent hazard of nucleic acid contamination. Nucleic acids from the previous rounds of purification could be carried over to the subsequent rounds there by compromising the quality of the purified nucleic acids. It is, therefore, imperative that any technique of column regeneration must be absolute, in other words, there should be no residual DNA left on the column after regeneration.

CNEC could be regenerated by a simple exposure to acid

We identified that the silica matrix is extraordinarily stable when exposed to mild acid for extended periods, even months at room temperature. This observation opened up an avenue to recycle CNEC. At low pH, nucleic acids are fragmented due to depurination and could be removed from the binding matrix when the columns are washed. An exposure of the columns to 1 N HCl efficiently regenerated the columns by removing bound nucleic acids. A single column can be regenerated and reused for 10-20 times without a loss in binding capacity (Figure-1, panel A) in the absence of any chances of carry-over contamination. The DNA or RNA obtained this way is completely free of contaminating DNA from previous use and can be used for transfection, sequencing, real-time PCR or any other molecular application. We have regenerated diverse columns from a large number of manufacturers. With exception of the gel-elution columns, we could regenerate all other kinds of columns with great success. Both DNA or RNA columns could be regenerated. We re-use all types of columns for up to 10-20 times.

How to regenerate the columns?

The basic theme of column regeneration is a simple one. Treat the columns with acid, wash off the acid and regenerate the columns by treating them with a suitable buffer. The specific protocol and specific volumes of acid and buffers and the length of the treatments might vary depending on what type of columns that you are handling. Below we recommend two different protocols for the Qiagen miniprep spin or gravity-flow columns used for plasmid DNA preparation. This will work as a guideline on how to regenerate other columns.

Spin columns:

1. After use, rinse the columns thoroughly with water to remove any bacterial or other debris.
2. Drop the columns in a beaker containing 0.1 or 1.0 N HCl and leave them for overnight (16 h) or longer.
1. One hour incubation is sufficient in 1 N HCl. But for 0.1 N HCl you must leave the columns for minimum 16 h.
2. You may leave the columns indefinitely in acid. This is advantageous since no microbial growth is possible in acid.
3. This treatment will completely destroy the DNA bound to the column.
4. You may regenerate the columns individually or in batches.
3. Rinse the columns thoroughly with autoclaved distilled water until all the acid is removed.
1. The number of washes and the volume of water used depend on the number of columns being regenerated.
2. Wash the columns 5 to 6 times until you are sure that all the acid is removed.
3. Use only autoclaved distilled water or an equivalent.
4. Rinse the columns a couple of times with TE, or once with Qiagen Buffer QBT or an equivalent. The columns are ready for a fresh application.

Gravity-flow columns: Qiagen Tip-20 columns.

How to store regenerated columns?

At times, you may want to regenerate a large number of columns for later use. The best strategy is to store the columns in 1 N HCl because the acid not only regenerates the columns but also prevents microbial growth on the columns. Thus the acid functions as a regenerative agent and as a preservative. You may however, try the following alternative protocol to store the regenerated columns outside acid.

How to make buffers?

Commercial kits supply limited quantities of buffers required for nucleic acid regeneration. If regenerating columns, you must make your own buffers. The compositions of various buffers are usually provided in the technical brochure supplied along with the kit. For instance, ‘Qiagen Plasmid Purification Handbook-2005’ has compositions for all the buffers in ‘Appendix-B’. The Handbook is available from the supplier’s Homepage. The table containing buffer composition is provided here.

Reference:

You will find more technical information and data on column regeneration in the following publication that is freely accessible from the journal website.

Nagadenahalli B. Siddappa, Appukuttan Avinash, Mohanram Venkatramanan, Udaykumar Ranga (2007). Regeneration of commercial nucleic acid extraction columns without the risk of carry-over contamination. Biotechniques 42: 186-192. http://www.biotechniques.com/article.asp?id=112327

Questions?

Udaykumar Ranga
JNCASR, Bangalore, India
udaykumar@jncasr.ac.in