Methods and reagents: Is RNase-free really RNase for free? 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 some contamination problems seen with the use of commercial RNase-free enzyme preparations, as well as a few other tips. For details on how to partake in the newsgroup, see the accompanying box. Free RNase ********** When using RNase-free DNase I to clean up an in vitro transcription reaction before RNase protection or competitive RT-PCR, netters have found that some products sold as `RNase-free' really are not. Recently, Michael Clark (michael-clark@uiowa.edu) posted a somewhat disturbing story about the use of an RNase-free reagent with which he was having difficulties. An enzyme preparation that was sold specifically for removing leftover DNA from RNA samples actually contained RNase activity. After losing several mg of purified RNA from reactions that previously did not show this activity, he tracked down the source of the contaminating RNase to a commercial enzyme preparation of RNase-free DNase I. Other netters warned that it is common for some companies to neglect doing any tests with their preparations and that it is always wise to run your own quality controls on any new commercial enzyme before risking any precious samples. Not only should companies do strict quality control, but they should also guarantee their preps to be free of RNase. Unfortunately most do not and implementing such a program might cause an increase in enzyme prices. On the brighter side, although it was not the best of advertising schemes, it appears that the commercial supplier who sold the contaminated enzyme had inadvertently given away a free sample of their co-purified RNase along with their DNase I prep. Media with an attitude ********************** Many researchers use extra rich 2xYT [1], superbroth, Terrific Broth [TM] from Life Technologies, Inc. or Circlegrow [TM] media from Bio101 to increase the yield of plasmid DNA from minipreps, or for purification of large amounts of overexpressed recombinant proteins from Escherichia coli. [2] Recently, one netter noticed that there are several different recipes for superbroth floating around and wondered if these really make a difference in yield as compared to the standard LB media used by most laboratories for growth of E. coli. One recipe for superbroth [3] calls for 33 g/l tryptone, 20 g/l yeast extract, 7.5 g/l NaCl, and 3.5 ml of 10 M NaOH per liter, while another recipe posted to methds-reagnts was 32 g/l tryptone, 20 g/l yeast extract, 5 g/l NaCl. Although they are of slightly different compositions and no study has been done to find which is best, either one should give better yield than LB media. One person wrote that instead of purchasing Terrific Broth [TM] (TB), it can be prepared in-house as 12 g/l tryptone, 24 g/l yeast extract, 0.4% glycerol and buffered with 17 mM KH2PO4 and 72 mM K2HPO4. As with superbroth, this media was found to be useful not only for plasmid preps, for which it was developed [3], but also proved to be excellent for protein isolations as well. In the introductory study [4], it was reported that the saturation density of E. coli DH5alpha grown in LB was only 1 x 10^8 cells/ml, whereas the same strain grown in TB gave a saturation density of 8 x 10^9 cells/ml and four to seven times more plasmid DNA. Moreover, netters recently discussed how one might push the cell density to the max, perhaps by providing much more aeration to the culture. One trick that Dave Smith (davesmith@bioch.tamu.edu) suggested in order to supply a sufficient amount of oxygen needed to optimize a bacterial culture is to force filtered air through an autoclavable air stone of the variety sold by pet or aquarium supply stores. The air hose can be connected to an in-house air supply or an aquarium pump while the flask is on the shaker or sitting in a water bath. It is not known if shaking is absolutely necessary given the amount of air flow, but the amount of aeration obtained from air stones was thought to be far better than if beveled culture flasks are shaken at maximum speed. This netter wrote that using forced air can push cells up to an OD550 of nearly 100 units and that this can give at least 20-fold higher yields of protein in some cases. When setting up such a system, however, caution should be taken not to clog the tubes. The stone and tubing should be autoclaved separately from the media and fitted into the flask afterwards, as caramelization can easily gum up the microporous grey pumice rock. With any amount of air bubbling through the media, an anti-foam emulsion, such as Antifoam A Emulsion obtained from Sigma Chemical, might become a necessity. Blots R Us ********** Marking your membrane filters for orientation with radioactive fountain pen ink before exposing them to X-ray film has been common practice for years, and is most likely a throwback from the days when only radioisotopes were used for tracking probes. Many cloning manuals still suggest mixing radioactive materials into your own ink. The drawbacks of doing this, however, are that the radioactivity has a limited life span, so your ink might not always give the best image after some time, and mixing 32-P with bottles of ink can be dangerous if not messy. Many people now do non-radioactive assays with chemilluminescence in order to avoid radioisotopes altogether. To give confused researchers the opportunity to figure out which end is up, some biotech companies sell stickers marked with their insignia stamped in fluorescent ink, marking pens impregnated with fluorescent ink, or fluorescent tape that can be used for blackening a small section of the film. Writing on the tape with a dark pen containing a dense black ink and then placing this next to the film will create a negative image of your own writing on the X-ray film. One problem with all this, however, is that these materials are generally very expensive. An inexpensive way to mark your filters is by using fluorescent paints or illuminescent Halloween stickers, which can be bought at your local toy or party store. Recently, one netter wrote that `Creative Touch' brand luminous acrylic paint (catalog number 628612) manufactured by Palmer Paint Products, Inc., Troy, MI, USA, can be found in many fabric, sewing or hobby stores, or other paints such as those used for fishing lures, can be bought from sports stores. A substantial dilution of the concentrated paint might be required for the right amount of blackening on film, but a small sample should cost far less than made-for-laboratory stickers and will last for a lifetime of glow-in-the-dark fun. Of course, these materials could be used in combination with radioactive assays to cut down on the wasted isotope and disposal problems. Elution Solution **************** Purification of plasmid DNA using silica based resins is now much more common than the older CsCl-ethidium bromide bouyant density centrifugation methods of the past. In addition, using silicates for recovery of DNA fragments from agarose gels with kit reagents or homemade resins has now become the standard practice. [5] Although many DNA isolation kits are flooding the market, each comes with a suitable buffer for the elution of the DNA from the silica matrix on which it was electrostatically bound. The instructions included with the different kits usually recommend the use of particular buffer, which is supplied, but the elution buffers from various kits can vary widely from one kit to another. In two previous Methods and reagents columns (TiBS 19, 182-183 and 19, 388-389), I discussed the recovery of plasmids or DNA restriction fragments that have been bound to silica particles and I wrote that using sterile distilled water and incubating tubes in a water bath heated to 65 degrees C during elution instead of using a Tris-EDTA (TE) buffer could increase DNA yield. Getting the DNA to come off the silica particles using water might not be the best way for maximum recovery, however, and it now appears that the efficiency of recovering a DNA fragment from silica could be significantly affected by its AT content. A recent study [6] showed that linear DNA fragments containing more than 62.5% AT were recovered poorly with water at 55 degrees C, while those of lower AT content were recovered efficiently at that temperature. Interestingly, a difference was observed when distilled water was used as compared to Milli-Q water, presumably because trace amounts of ions remaining in the distilled water allowed for a slight increase in melting temperature. Not only did this suggest that low ionic conditions are preferable to Milli-Q water for the elution step, but it also implied that recovering DNA fragments with water at temperatures higher than 55 degrees C without prior knowledge of the AT content could be risky. The study suggests that a better overall elution buffer would be 0.1xTE buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) containing 10 mM NaCl. Alternatively, the recovery of covalently closed circular plasmid DNA was not affected by elution with plain old water and it is therefore not necessary to alter your plasmid mini-prep protocol based on these findings. References ********** [1] Messing, J. (1983) Methods Enzymol. 101, 10-89 [2] San, K. Y. et al. (1994) Ann. NY Acad. Sci. 721, 268-276 [3] Davis, R. W., Botstein, D. and Roth, J. R. (1980) A Manual for Genetic Engineering. Advanced Bacterial Genetics, p. 202, Cold Spring Harbor Laboratory [4] Tartof, K. D. and Hobbs, C. A. (1987) BRL Focus 9(2), 12 [5] Hengen, P. N. (1995) in Molecular Biology: Current Innovations and Future Trends, (Griffin, A. M. and Griffin, H. G., eds.), pp. 39-50, Horizon Scientific Press [6] Kaur, R., Kumar, R. and Bachhawat, A. K. (1995) Nucleic Acids Res. 23, 4932-4933 ******************************************************************************* 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/mar96.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1996. Methods and reagents - Is RNase-free really RNase for free? Trends in Biochemical Sciences 21(3):112-113. ******************************************************************************* * 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 /--------------------------/* *******************************************************************************