Millipore Technical Publications
Effect of Centrifugal Ultrafiltration on Large Fragment DNA Integrity | Protocol - WWW-UF > Product Catalogue | |
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Introduction
Centrifugal ultrafiltration provides a fast and easy method for the concentration and desalting of biological molecules. Previous reports document the use of ultrafiltration for desalting samples of nucleic acids1, removing excess primers from PCR2 reactions3, or concentrating DNA/RNA samples without the need to use ethanol precipitation4.Centricon and Microcon filters offer a convenient, reproducible means for centrifugal ultrafiltration of samples from 50 µL to 2 mL. They insure high sample recovery with their patented inverted recovery spin.
The analysis of complex genomes depends on the ability of the researcher to prepare pure, high molecular weight DNA. When preparing high molecular weight DNA samples for cosmid cloning or other applications, it is important to treat the DNA gently to avoid shearing or other damage to the sample. As virtually all protocols for the preparation of high molecular weight DNA require, at some point, buffer exchange and/or concentration, centrifugal ultrafiltration can provide an efficient alternative to standard procedures.
To be useful, centrifugation must not cause breakage of the DNA during the ultrafiltration procedure. This article summarizes the results of a series of experiments designed to evaluate the effect of g forces on various samples of high molecular weight DNA during centrifugal ultrafiltration in Microcon or Centricon units.
Methods
DNA LadderAs a model system to check for the introduction of single-strand nicks during the ultrafiltration spin, supercoiled Ladder DNA (Gibco-BRL, Gaithersburg, MD) which ranges in size from 2 to 8 kb was used. DNA diluted with TE buffer was loaded into Microcon 30K NMWL and Centricon 30K NMWL devices. The Microcon units were spun for 7 minutes at 12,000 x g, and the Centricon units were spun for 30 minutes at 5,000 x g. The concentrated DNA sample (retentate) was diluted again with TE buffer (to 500 µL or 2 mL, depending on the device used) and spun once more, as described above. The dilution step was repeated a third time. After the third concentration spin, the retentate was collected by placing the sample reservoirs upside down in new vials and spinning the units for 1 minute at 1,000 x g.
The concentrated DNA was run on a 0.9% SeaKem® GTG agarose gel (FMC) and stained with ethidium bromide to monitor sample integrity (Figure 1.1). DNA bands in lane 1 (starting material) are indistinguishable from the bands on lanes 2–4, which were spun repeatedly in the ultrafiltration devices. There is no evidence that any supercoiled DNA was converted to the relaxed or linear forms during the concentration procedure, which would be the case had single-strand nicks been introduced during centrifugation.
Figure 1.2 Supercoiled DNA Ladder.
Discrete Size Plasmids
The next set of experiments monitored the effect of centrifugal ultrafiltration on single population, discrete size plasmids. The plasmids used were pBR322 (4,361 bp; New England Biolabs, Inc.), pSPT18 (3,104 bp; Boehringer Mannheim) and pXTl (10,400 bp; Stratagene). Samples (1 µg) of each plasmid were spun in Microcon 30K NMWL or Centricon 30K NMWL units, as described previously. The starting material and retentates were run on a 1% agarose gel and stained with ethidium bromide.
The results are shown in Figure 1.2. As in the case of the DNA Ladder, the concentrated samples (lanes 2, 3, 5, 6, 8, and 9) appear to be identical with their corresponding starting material. Again, there is no evidence of conversion of the supercoiled form to the relaxed form after exposure to g forces of 5,000 x g for 90 minutes and 12,000 x g for 21 minutes during the concentration spins.
| Figure 1.2 Specific plasmid DNA | | |
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To monitor the integrity of molecules in the size range of genomic DNA after centrifugation, lambda DNA (49 kb; Boehringer Mannheim, Indianapolis, IN) and Bsu36 l digested BacPAK6 DNA (125 kb) was used. The samples were diluted and centrifuged as described above. The retentates and starting material were run on a 1% agarose gel in a CHEF-DR® II pulsed field electrophoresis system (Bio-Rad, Richmond, CA). Electrophoresis was performed at 200 V at 14 °C in 0.5X TBE with ramped pulse from 1 to 6 seconds over 14 hours.
The results with the lambda DNA mimic those of the other samples run in these sets of experiments. No adverse effects are noted after spinning the DNA at g forces up to 12,000 x g in the ultrafiltration units. However, the larger BacPAK®6 DNA does show some degradation after ultrafiltration at both 5,000 and 12,000 x g (Figure 1.3, smearing in lanes 5 and 6). Although a large percentage of the sample appears to be intact, there was loss of integrity of the BacPAK6 DNA sample after the concentration procedure.
Figure 1.3 Large fragment DNA
Conclusions
DNA samples of up to 49 kb were concentrated repeatedly without any loss of sample integrity. Some loss of integrity was observed with a 125 kb sample, although it was not complete and represents a small percentage of the total DNA in the sample. For large fragments of DNA, centrifugal ultrafiltration provides a fast and efficient method to concentrate or desalt the sample. It results in high recovery of intact product.References
- Takagi S, Kimura M, Katsuki M. BioTechniques 1993;14(2):218–21.
- PCR is covered by U.S. patents issued to Hoffmann-LaRoche, Inc.
- Sheng N, Zhang J, Whitton JL, McKee T. BioTechniques 1993;14(5):781–4.
- Ruano G, Pagliaro EM, Schwartz TR, Lamy K, Messina D, Gaensslen RE, Lee HC. BioTechniques 1992;13(2):266–74.
