Methods and reagents: Caring for your hybridization membranes 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 time limitation for using nylon-based and nitrocellulose membranes for Southern hybridizations, and there are a few other helpful hints. For details on how to partake in the newsgroup, see the accompanying box. A recent discussion revealed that some nitrocellulose membranes used for Southern hybridizations might be unstable when stored for extended periods. Netters have noticed that, after some time, they give low or uneven transfer efficiencies when DNA is blotted onto them. The most recent discussion about membranes used for Southern transfers centered on the supported nitrocellulose membranes sold by Schleicher and Schuell (S&S). S&S recently changed the name of their BA-S 85 [TM] membranes to the more sleek, stylish name Optitran [TM], perhaps suggesting that a sports car purchase is in the works from grant money, rather than a box of transfer membranes. The filters come as standard with the patented TurboBlotter [TM] transfer system, and with S&S's a vacuum intake manifold - optionally equipped with four on the floor (skid-proof rubber feet to prevent lab benchmarks, that is). Warranty notwithstanding, blotting membranes from other companies may also have a limited lifespan and are equally susceptible to decay. Unfortunately, when the membranes in hand do not work, some researchers tend to distrust the brand name, and switch suppliers. Packages of membranes are rarely date-stamped by their manufacturers, and workers seldom make a note of the date on which they opened a new package. Membranes might not be used for years and can be passed from worker to worker, without anyone knowing exactly how long the package has been opened. Furthermore, when the time comes for the membranes to be used for an experiment, it can be quite difficult to spot a bad batch of membranes, and might be a big risk to assume that they are still good. But throwing out a good set could be costly. Nitrocellulose membranes designed for blotting usually contain a humectant to make them wettable, and storage under humid conditions will cause the membranes to absorb water. Water molecules from the air will eventually occupy the hydrophilic sites required for binding DNA and will therefore change the blotting characteristics of the membrane. Some netters wrote that membranes that have gone off sometimes look different from freshly opened ones: the bad membranes have a rippled or corrugated appearance. However, this test can be subjective, and wrinkles on the edges surely are not the most clear-cut evidence for a bad lot. Netters agree that a good way to tell whether a membrane has gone off is by wetting it in a puddle of water or transfer buffer. If it doesn't wet immediately and uniformly as you lay it into the water, the membrane is unlikely to transfer DNA evenly when used for capillary blotting from an agarose or polyacrylamide gel. Someone suggested that the membranes should be usable for years once opened and, although it isn't recommended specifically by the supplier, the membranes might be better off stored under desiccation or within an area where they are protected from the atmosphere and laboratory chemicals. However, storage in a desiccator could be a disaster, since the membranes might become brittle. A relative humidity of 50-60% and a constant temperature of approximately 15-20 degrees C were suggested as better long-term storage conditions. (Precious membranes could be treated like a fine cigar and tucked safely away in a humidor). Most netters feel that any membrane stored on the shelf for more than a year without adequate protection should be suspected as bad and, resources permitting, should be replaced. Report card on polymerases ************************** In a recent Methods and reagents column (TIBS 20, 85-86), a method of cloning products of the polymerase chain reaction (PCR) was discussed that involves the addition of deoxyadenosine residues to the 3'-end of PCR products by Taq polymerase. In that article, I reported that low-efficiency cloning of PCR products into T-vectors could result from degradation of the T-overhangs on the vector, perhaps by contaminating exonucleases. Taq polymerase is not the only enzyme used for amplification of DNA by PCR, and removal of T-overhangs from the vector DNA might not be entirely to blame for a blown TA cloning attempt. Since the efficiency of nucleotide addition to the 3'-end of DNA fragments varies among different enzymes, depending upon which nucleotide is on the 3'-end of the fragment and which DNA polymerase is used, low-efficiency TA cloning could be due to the fact that even the smartest PCR products don't always get their As. One school of thought is that a low-efficiency cloning could be due to deoxyadenosine residues not being added to the PCR product during the extension reaction, especially if a combination of polymerases with proof-reading ability is used for the production of long amplicons. Some enzymes, such as Pfu polymerase, have been shown to remove any extra bases on the 3'-end of the amplicon. [1,2] Picky, picky! ************* In last month's Methods and reagents column (TIBS 20, 124-125), the reamplification of a PCR product was discussed in detail. One of the methods for recovering a DNA band for a secondary amplification is the use of a toothpick for stabbing the band. Although it was not spelled out in the article, it was assumed that the toothpick would be wooden. A recent study [3] revealed that wooden toothpicks contain a water-soluble inhibitor of amplification of DNA by Taq polymerase. Interestingly, the inhibitor is probably intrinsic to the wood, not introduced by the manufacturing process. Although it was not discussed in the paper [3], and it is unknown how it could survive the milling and packaging processes, the contaminant could be a toxin of sorts or, oddly enough, genomic DNA from the white birch trees used for making the picks. Whatever the substance is, it could account for many, many failed PCR experiments. Based on this new information, netters are now recommending the use of plastic toothpicks or pipette tips for band stabbings. More sticky business ********************* Tricks for keeping your polyacrylamide gel from sticking to the glass gel-plates include the use of a silanizing agent such as Rain-X [TM] used to treat automobile windshields, PAM [TM] spray-can vegetable oil, or Pledge [TM] furniture polish. Each of these can be applied by simply spraying directly onto the glass plate and wiping it with a laboratory towelette each time the plate is used. If only one plate is treated, the gel will stick to the untreated one when the plates are split - and the used towelette can be used for doing up the rest of the lab bench before you throw it out. References [1] Hu, G. (1993) DNA Cell Biol. 12, 763-770 [2] Costa, G. L. and Weiner, M. P. (1994) Nucleic Acids Res. 22, 2423 [3] Lee, A. B. and Cooper, T. A. (1995) BioTechniques 18, 225-226 ******************************************************************************* 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/apr95.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1995. Methods and reagents - Caring for your hybridization membranes. Trends in Biochemical Sciences 20(4):160-161. ******************************************************************************* * 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 /--------------------------/* *******************************************************************************