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Cole-Parmer Technical Library

Rapid Downward Southern Transfer Using the S&S TurboBlotter System

Reprinted with permission of Schleicher & Schuell, Inc.

In standard southern and northern blotting procedures, capillary forces cause an upward migration of transfer solution through the agarose gel to the absorbent layer of paper, facilitating the elution of nucleic acids from agarose gels to an immobilizing matrix. A common problem associated with capillary transfers is gel flattening, caused by the weight of gel blot paper on top of the gel and also by a weight that is usually added to the top of the entire stack. Gel flattening can result in poor transfer of the DNA or RNA samples to hybridization membranes.

To eliminate gel flattening and inferior results, a downward capillary transfer was developed (Lichtenstein, et al., 1990). Shortly thereafter, a modified version of the downward transfer was described (Chomczynski, 1992), which substituted an alkaline solution for the SSC buffer originally used, increasing the hybridization efficiency and dramatically decreasing the time needed for transfer. Downward alkaline southern transfers can be done in one hour, which is a substantial reduction of blotting time compared to standard overnight transfer techniques.

Figure A
Duplicate southern blots—Chemiluminescent detection: An EcoRI digest of pUC18/Vimentin, separated on a 1% agarose /TBE gel, alkaline denatured and transferred to Nytran SuPerCharge using the TurboBlotter. The membranes were hybridized overnight, washed, blocked and incubated with substrate. The membrane was exposed for 30 minutes at 37°C.

In order to make the downward alkaline transfer simpler and more efficient, S&S developed the TurboBlotter system for setting up downward transfers. This device is designed to hold the appropriate blotting papers and agarose gel in a format where the buffer wick can be placed at a level even with the surrounding buffer tray. This arrangement allows wicking of buffer from two sides of the gel rather than one as originally described, resulting in a more efficient transfer. The TurboBlotter uses S&S GB002 and GB004 thin and thick blotter paper, which provide excellent wicking characteristics and guarantee consistent results from blot to blot. Blotter packs provided with the TurboBlotter system include pre-cut, pre-stacked GB002 and GB004 paper, as well as a pre-cut buffer wick.

The TurboBlotter now includes the new Nytran SuPerCharge nylon membrane for high resolution blots (see Figure A). This membrane is manufactured using a new technology and features the highest positive charge available in a nylon membrane, very low background, and consistent membrane morphology.

The Nytran SuPerCharge TurboBlotter System provides a rapid, inexpensive blotting kit for nucleic acid blots with high resolution and low background. Better blots are achieved in one hour with a simple set-up that eliminates the mess of standard upward transfers.

Neutral southern & northern blots can also be performed in the TurboBlotter system using SSC transfer buffer. Neutral transfers take three hours, which is still a reduction in blotting time compared to overnight.


References

  1. Chomczynski, P. 1992. Anal. Biochem. 201:134-139.
  2. Lichtenstein, A.S., V. Moiseev, and M.M. Zaboikin. 1990. Anal. Biochem. 191:187-191.


Photos courtesy of Schleicher & Schuell, Inc.
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