For solution volumes less than ~100 |l, the use of dialysis membrane as described above can result in unacceptable sample loss. The method described below can easily dialyze volumes as small as 10 |l. The sample is held in a 0.5-ml microcentrifuge tube, with dialysis membrane covering the open end of the tube.
Additional Materials (also see Basic Protocol 1)
0.5-ml microcentrifuge tube Pasteur pipet Cork borer
1. Puncture the lid of each microcentrifuge tube using a heated glass Pasteur pipet (wide end heated) or cork borer to completely remove the center part of the lid (see Fig. A.3H.1). Open lid and place a single layer of dialysis tubing over top of tube, then close lid to hold dialysis tubing in place.
The success of this protocol is dependent on a tight-fitting cap, so make sure the cap is well-seated. On the other hand, an excessively tight-fitting cap can rip the membrane when it is inserted. Sarstedt tubes work well.
Alternatively, some researchers use a microcentrifuge tube without a cap. The sample is placed in the microcentrifuge tube, a dialysis membrane is placed over the top of the tube, and the membrane is secured with a rubber band. With this method, however, there is a risk that a small sample—e.g., 10 to 20 pl—may be lost around the edge of the tube.
2. Turn tube upside down and shake reaction mixture onto the membrane surface. Tape each tube, dialysis surface down, to the side of a beaker, then fill the beaker with PBS. Dialyze 2 hr at 4°C with stirring.
Alternatively, place the tube in a styrofoam or foam sheet (e.g., foam microcentrifuge rack, Fisher) then place, with the tube inverted, on the surface of the dialysis buffer.
Make sure the dialysis surface is in contact with the buffer and that no bubbles exist. This can be accomplished by gently centrifuging the tube in an inverted position in a tabletop centrifuge.
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