Methods and reagents: ...On the magic of mini-preps Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses the problems encountered by some people when switching from Promega's Magic [TM] mini-prep plasmid DNA isolation columns to their new Wizard [TM] mini-prep kits. For details on how to partake in the newsgroup, see the accompanying box. Some of the dedicated users of Promega's Magic [TM] mini-prep plasmid DNA isolation columns have recently been quite disgruntled, since Promega has changed the components of the kit to make way for the new Wizard [TM] mini-prep kits which contain a different proprietary binding resin. Netters have always been impressed with the speed of the Magic [TM] mini-prep kit, but some have now grown wary of the Wizard [TM] version since they have experienced intermittent problems when changing over to the new kit. The new proprietary resin ************************* Unfortunately, researchers who have become accustomed to the Magic [TM] version of the kit have tweaked their experiments for maximum yield and purity of DNA and are now faced with altered conditions and loss of consistency in their research when switching to the Wizard [TM] version. Since the resin is an unknown reagent, scientists are concerned over the switch because they can no longer repeat their previous `Magic' experiments using the `new and improved' resin. The complaints are that the DNA isolated is resistant to restriction-enzyme digestion and, when run on an agarose gel, the plasmid DNA appears to be nonspecifically degraded, perhaps by contaminating nucleases in the cell lysates or within the mini-prep binding resin. Upon purifying the DNA by removal from an agarose gel and phenol extraction, the DNA may still be resistant to restriction enzymes. Are the perceived problems due to incidental differences in the handling of the kit components among different laboratories, changes in the binding solutions, which contain either guanidine thiocyanate or guanidine hydrochloride, or the result of the new resin as the binding matrix? Recommendations *************** Promega's Technical Service is aware of the complaints associated with the kit and has found that storage conditions for the binding solution can be variable after the kits are shipped. Under some circumstances, the binding solution containing guanidine hydrochloride might be exposed to temperatures below the precipitation point, causing it to flocculate. They recommend that, if precipitates are seen within the bottle, the solution should be warmed by incubation at 37 degrees C for at least ten minutes and then cooled to room temperature (<30 degrees C) before use. Some netters have said that this precaution does not alleviate the poor results obtained in their hands. Methodology problems ******************** One of the reasons for the problems people are having could be inherent to the method itself regardless of the source of the resin. Some people thought that bad preps could be the result of incomplete drying of the resin before elution of DNA. Residual ethanol still wetting the resin could contaminate the sample if co-eluted with it. This may be especially true for the larger-scale kits, which have been reported to yield DNA resistant to restriction-enzyme digestion no matter how much enzyme is used. One way to avoid this with the mini-prep system would be to increase the ethanol concentration within the wash solution from 50% to 55% and centrifuge the plastic Luer-Lock-fitted mini-columns within 1.5 ml microcentrifuge tubes and/or completely vacuum dry the DNA-resin complex. The larger syringe types could be centrifuged for a few minutes at about 1000 RPM in a swinging-bucket rotor, dried on the Promega Vac-Man [TM] vacuum manifold, or fitted with a large-bore needle and lanced through a stopper within a side-arm flask. An aspirator is also recommended as a better source for drawing a vacuum than an in-house vacuum line because the latter may not be strong enough to fully dry the resin. Another idea is that a more forceful centrifugation during the elution step could allow some of the resin to pass from the column and contaminate the DNA. It was also thought that perhaps those who have had few troubles with the method are eluting the DNA with distilled water heated to 65 degrees C as opposed to using Tris EDTA solutions. Since an elution buffer is not supplied with the kit, researchers could assume that the TE buffer recipe calls for 25 mM Tris and 10 mM EDTA. Promega recommends that if a Tris EDTA buffer be used, it should contain 10 mM Tris and 1 mM EDTA, and that higher concentrations of EDTA could be squelching out Mg2+ ions necessary for restriction-enzyme activity. On the other hand, the effect of excess EDTA could be insignificant because, although it is not necessary to protect DNA in this way if stored frozen, buffered EDTA solutions have been used for storage of DNA samples for many years without deterring subsequent enzymatic restriction digests. Others have had no problem with the new kits, reporting that they work just as well as the older version. The DNA was digestible with various enzymes and no problems were experienced when using the purified DNA for automated fluorescent sequencing. Additionally, template amplified by the polymerase chain reaction from plasmid DNA isolated using the kit is working for manual and automated sequencing reactions. Multiple side-by-side comparisons of the Magic [TM] kit versus the Wizard [TM] kit using aliquots of the same culture of DH5alpha carrying a derivative of pBR322 and taking the precautions mentioned above were performed by the author; no difference in yield or purity of DNA was seen. Home-made kits ************** Many biotechnology companies now sell mini-prep kits with secret proprietary resins. As an alternative to costly kits, netters have have been making home-made versions of binding resin for some time by using crushed flint glass from scintillation vials [1], diatomaceous earth [2], Celite [R], or silica particles from other sources [3-5]. Makeshift columns can be constructed using a simple syringe cartridge and Whatman [R] 3MM filter paper, or microcentrifuge tubes stuffed with siliconized glass filters. Most claim that the results with these are comparable to those purchased. Since the plastic gizmos supplied with any of the kits on the market can be costly, they can be reused for some types of experiments by first boiling in denaturing solution and then autoclaving to destroy previous DNA contaminants. Of course, reusing the columns is not recommended by any manufacturer. Together, the cost of a plasmid preparation using this method can be reduced to a fraction of what is charged for the kits, although the cost in research time in setting up such homemade systems must be considered. References: [1] Vogelstein, B., and Gillespie, D. (1979) Proc. Natl. Acad. Sci. USA 76, 615-619 [2] Carter, M. J., and Milton, I. D. (1993) Nucleic Acids Res. 21, 1044 [3] Marko, M. A. et al. (1982) Anal. Biochem. 121, 382-387 [4] Sparks, R. B. and Elder, J. H. (1983) Anal. Biochem. 135, 345-348 [5] Willis, E. H. et al. (1990) BioTechniques 9, 92-99 ******************************************************************************* Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health. Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author. You found this at the World Wide Web (WWW) Uniform Resource Locator (URL) ftp://ftp.ncifcrf.gov/pub/methods/TIBS/apr94.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1994. Methods and reagents - ...On the magic of mini-preps Trends in Biochemical Sciences 19(4):182-183. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* ******************************************************************************* ******************************************************************************* *** Promega Replies *** Last September, Promega introduced a new formulation to replace the Magic [TM] resin in our DNA purification systems. One component of the Magic [TM] resin was diatomaceous earth, a material that we found to be unacceptably variable in quality and composition, often containing dirt and other contaminants. We were unable, in good conscience, to plan the future of this product line on a component that was so uncontrollable. Selection and design of a new formulation took more than a year of intense effort. Before introduction, the Wizard [TM] resin was rigorously tested in all major molecular biology applications - restriction digestion, sequencing, labeling, transcription, etc. - in which it performed well and reproducibly (and contained no dirt!). In addition, we found that the Wizard [TM] resin increased maxiprep yields significantly. Despite the extensive testing, we knew we could not anticipate all the circumstances in which Wizard [TM] products would be used, and that some fraction of customers would find the resin change problematic. We were pleased to find that the problems were, in fact, very limited: most users expressed complete satisfaction with the new Wizard [TM] formulation. We are, none the less, concerned about the difficulties any user may experience. Customers are strongly urged to call Promega's Technical Services staff, who are eager to help resolve any issues that may arise in adapting protocols to the new resin formulation. It is our policy to respond to every concern and pursue it until solved. Over the past couple of months, user input has guided us to make substantive improvements to the protocols and has shown us where new improvements might be made. Promega is completely committed to the Wizard [TM] product line and is confident that all users will find Wizard [TM] products as valuable as Magic [TM], if not more so. GUY PAGE Promega Corporation 2800 Woods Hollow Road Madison, WI 53711-5399 USA *******************************************************************************