Cellu·Sep H1 tubular dialysis membranes are designed for sensitive dialysis applications where glycerol, sulfur compounds, or small amounts of heavy metals will interfere with subsequent steps. With the recent expansion of molecular biology and the consequent need to desalt nucleic acid (DNA and RNA) samples, the characterization of membranes as a function of their behavior with DNA/RNA and the need for highly clean membranes have become important factors.
Cellu·Sep H1 membranes are pre-treated and packaged wet in a preservative solution to prevent microbiological growth. H1 membranes are prepared in a wide range of flat widths and MWCOs. Individual-use 50 cm lengths in plastic vials help avoid waste, contamination and restorage problems. H1 is also available in continuous 10m and 5m rolls.
Membranes are characterized by Molecular Weight Cut-off (MWCO), which relates to the expected membrane retention of proteins according to their molecular weights. Although some membrane characterization involves the use of linear (natural or synthetic) polymers of discrete sizes rather than native proteins, the initial estimation of a membrane's ability to purify or desalt a particular protein continues to depend on the MW of the protein in question. Ultimately, however, the size and the three-dimensional shape of a molecule determines whether or not it will pass through a membrane. MWCO has become a convenient guide for determining which membrane to use for specific applications.
Nucleic acids are routinely characterized by their length in bases (single stranded-ss) or base pairs (double stranded-ds) and they display far less tertiary structure than proteins. Although one can easily convert from length to the approximate MW of a nucleic acid sample, we felt that a direct comparison of nominal membrane MWCO to retention of various lengths of DNA (ss and ds) would be useful.
|
|
Both the 2,000 and 5,000 MWCO membranes can be used to dialyze any size fragment (ss or ds). However, 10,000, 15,000 and 25,000 provide a faster alternative when dialyzing double-stranded samples.
The data collected to date indicate that Cellu·Sep H1 membranes provide an efficient method for the purification of DNA/RNA samples. Studies comparing membrane MWCO to length of ss or ds DNA indicate that sample behavior is similar to that of proteins: it is size dependent. Choosing the appropriate membrane according to its retention of DNA (by length), provides a superior alternative to using the MW of the DNA fragment.
1. Remove as many H1 membranes from the vial as needed. Cool store the unused membranes at 4-8 °C.
NOTE: Wet membranes are most susceptible to microbes and fungi. It is vital that any excess membrane be stored with satisfactory protection. Click here for additional preservative buffer suggestions.
2. Remove membrane from its vial and rinse with distilled water. Cut membrane to desired length and securely clamp one end.
3. Check integrity of tubing and clamp by filling the membrane with water or dialysis buffer, holding the unclamped end closed.
4. Pour out test solution and load sample. (For concentrated salt samples, leave space in the tubing to allow for net flow of water into the sample and to prevent tubing from bursting.)
5. Immerse dialysis tubing in beaker or flask containing a large volume (usually 100 to 1000-fold that of the sample) of the desired buffer and dialyze for several hours at the desired temperature with gentle stirring.
NOTE: Low-molecular weight salts and buffers equilibrate within 3 hours with stirring. Equilibration times for viscous samples will be longer.
6. Change dialysis buffer as necessary. Usually two dialysis buffer changes are sufficient. When CsCl is removed from equilibrium density gradient-banded DNA, two equilibrations against a 1000-fold volume excess of buffer will decrease CsCl concentra- tion 106-fold, to a still significant 5 µM, and it may be necessary to change the buffer a third time.
7. Remove dialysis tubing from buffer, remove clamp from one end, and remove sample with a pipet.