Methods and reagents: Eliminating ghost bands from plasmid 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 updates the discussion of a ghost DNA band that continues to haunt netters. For details on how to partake in the newsgroup, see the accompanying box. In a previous Methods and reagents column [TiBS 19, 139-141 (1994)], netters complained about a ghostly plasmid band that appeared on ethidium bromide- stained agarose gels at a position below that of cccDNA, after pBluescript DNA had been isolated using a mini-prep kit. At that time, the exact source of the ghost band was unknown. The most striking feature of that contaminating plasmid is that it is resistant to restriction enzymes that normally cleave within the multiple cloning region of the vector DNA. Speculations concerning its origin included the host strain, the vector and the mini-prep kit used to isolate the DNA. Ghostbusters ************ Recently, Jon R. Sayers (j.r.sayers@sheffield.ac.uk) showed that the ghost band, affectionately known as Freddy, is not the result of the Escherichia coli strain XL1-blue used as host, but that the enzyme-resistant ghost is most likely formed because of alkaline lysis of bacteria. At first, he thought the band might be a replication intermediate, as suggested earlier, but it became apparent that the intensity of the ghost band varied depending on how the plasmid was isolated. Some workers could produce more ghost band than others when using an alkaline-lysis step in their protocol. The longer the exposure to alkali or the higher the incubation temperature during lysis, the stronger the ghost band appeared. After observing a similar ghost band in preparations of pUC DNA and some other non-phagemid vectors, Dr Sayers became sceptical of the proposal that the band was somehow due to the f1 origin present in the pBluescript vector, as was thought by other netters. He also observed that the faint ghost band disappeared when plasmid DNA, isolated using the Qiagen maxi-prep kit, was used as substrate in reactions with T5 exonuclease, which was done as a test for contaminating endonuclease activity in batches of purified T5 exonuclease. As the ghost band comprises approximately 3% of the total plasmid DNA prepared by the alkaline-lysis method, and it can transform competent E. coli cells, it is a source of background in transformation experiments that cannot be eliminated by overdigestion of the plasmid with restriction enzymes. This can be of special concern when genomic libraries are being constructed, because it can increase the number of non-recombinant transformants. Having dubbed himself the new plasmid ghostbuster, Dr Sayers set out to eliminate the ghost from plasmid mini-preps using the T5 exonuclease. He recently showed that treatment with T5 exonuclease, which has single-strand-specific endonuclease as well as 5'-exonuclease activity [1], hydrolyzed the ghost plasmid and reduced the transformation efficiency of the ghost by two orders of magnitude [2]. That the ghost plasmid can be digested with the nuclease implies that there are regions of single-stranded DNA or nicks present, as was observed earlier by netters. However, as the ghost band migrates further in the gel than cccDNA, it seemed unlikely to be the nicked, open circular form of the plasmid. Owing to these properties, the ghost band is now thought to be double-stranded cyclic, coiled DNA composed of two intertwined, but permanently denatured, single-strands of plasmid DNA. Interestingly, a mini-prep method not based on alkaline-lysis, such as a lithium chloride/phenol chloroform technique [3] or a boiling lysis method [4-6] alleviates the need to digest away the Freddy plasmid with T5 exonuclease, because these mini-prep methods produce little or no ghost band. The trade-off is, however, that increased contaminating sheared chromosomal DNA can be formed by these methods. One other option to rid plasmid preps of Freddy without the use of T5 exonuclease is to slice the restricted linear vector DNA band that has been separated from the ghost band by electrophoresis directly out of an agarose gel for library constructions. The drawback is that this will reduce the overall yield of vector DNA. Animosity toward viscosity ************************** Ever get frustrated with thick and messy solutions that just cannot be measured accurately with a micropipet? Some people say that using a graduated cylinder for measuring out viscous chemicals such as Tween-20, Nonidet P-40, glycerol or dextran sulfate, is not only inaccurate, but it is simply wasteful when smaller amounts of are to be measured. Some say that the liquid can be diluted with water to 20-50% w/v first and then this can more easily be measured out by pipetting to make more dilute solutions. However, the viscous chemical still needs to be measured out for this. Instead, netters suggest using a wide-bore or cut-off pipet tip, or a syringe to dispense solutions. A 1.0 ml or 5.0 ml tuberculin syringe without a needle, or one with a wider, 18-gauge needle attached, is perfect for measuring out glycerol or mineral oil. If the viscous material is added while the solution to be made is on a top-loading balance, the difference in weight can be determined for precise measurements, and netters say that it is much easier to control the flow of liquid with a syringe plunger than with a pipet. Warming the filled syringe in a microwave oven (without the needle of course) for a few seconds can also make it much easier to squeeze the liquid out. Cheap vacuum pump ***************** Robert Passey (r.passey@unsw.edu.au) recently wrote about how he built a very simple and cheap apparatus that generates and measures a partial vacuum suitable for vacuum blotting gels and slot/dot blots. The pump is essentially a 25 litre aspirator drum filled with water and is connected from its top to a blotting manifold. As water attempts to move out of the drum it generates a partial vacuum proportional to the difference in height between the upper water level and the outlet (e.g. 40 cm). Unfortunately, the vacuum is not strong enough to completely dry a full size sequencing gel. However, if for some reason you cannot afford a vacuum pump, are willing to spring for the cost of the drum needed, have plenty of room for the device in your lab or basement, are patient enough to fill the container, and are not picky about the decor, it might be worth the minimal effort to build one. Currently scheduled to be published in the May 1997 issue of BioTechniques, the details on how to make your own homemade vacuum pump are now available at ftp://sirronald.wustl.edu/pub/mbmiller/vacuum.htm References ********** [1] Ceska, T. A., Sayers, J. R., Stier, G. and Suck, D. (1996) Nature 382,90-93 [2] Sayers, J. R., Evans, D. and Thomson, J. B. (1996) Anal. Biochem. 241,186-189 [3] He, M., Wilde, A. and Kaderbhai, M. A. (1990) Nucleic Acids Res. 18,1660 [4] Holmes, D. S. and Quigley, M. (1981) Anal. Biochem. 114,193-197 [5] Sparks, R. B. and Elder, J. H. (1983) Anal. Biochem. 135,345-348 [6] Gomez-Marquez, J., Freire, M. and Segade, F. (1987) Gene 54,255-259 ******************************************************************************* 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/nov96.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1996. Methods and reagents: Eliminating ghost bands from plasmid preps. Trends in Biochemical Sciences 21(11):441-442. ******************************************************************************* * 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 /--------------------------/* *******************************************************************************