Methods and reagents: Protecting vector DNA from UV light 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 how to squeeze more life out of DNA isolated from agarose gels. For details on how to partake in the newsgroup, see the accompanying box. Sunscreen for your vector ************************* DNA that has been treated with ethidium bromide and exposed to UV light for only a few minutes before being excised from an agarose gel might not always result in an efficient cloning experiment. It has been known for some time that gel-purified DNA can be damaged by UV light during the isolation procedure and this can significantly lower the transformation efficiency of vector DNA [1]. One netter pointed out a recent study [2] showing that DNA fragments isolated from agarose gels are more efficient in bacterial transformations, in in vitro transcription assays and the PCR, if protected by 1-10 mM guanosine or cytidine while being electrophoretically separated. In that study, gel-purified DNA was shown to be damaged if exposed to near 300 nm wavelength UV light for as little as 20-45 s, but that the DNA can be protected by including blocking agents directly to the TAE electrophoresis buffer. Interestingly, the other nucleosides adenosine and uridine, were not found to be effective in thwarting the damaging UV rays. In addition, other compounds tested such as 1 mM phosphate or 5 mM DTT did not offer any protection either. Some netters are now trying to optimize their experiments by adding the UV protectant to their gels, and are now wondering if that same might be true for TBE-buffered gels, as no one yet knows if the protection is caused by a unique combination of buffer components or by the presence of guanosine. The question as to how exactly the nucleosides can afford protection to the DNA also remains, as it is not known whether the added components can somehow block the UV light from interacting with the DNA, or that they somehow aid in the rapid repair or photoactivation of exposed DNA within the gel. In any case, it appears that inhibitors of ligase, previously thought to be caused by impurities within agarose gels, might not be completely to blame for many low-efficiency cloning experiments. Doin' the Horizontal Blot ************************* Recently, there was a discussion on the options for semi-dry blotting of proteins for western blots. To perform westerns with proteins, or Southerns and northerns with DNA and RNA, macromolecules are separated within polyacrylamide gels, then transferred to and immobilized on nitrocellulose or nylon membranes for probing. Although efficient transfer of DNA has been reported to occur by direct contact between the gel and membrane without using any electro-blotting device [3], DNA fragments are typically transferred in the same fashion as proteins. Usually the gel, membrane and wetted blotting paper are sandwiched together between two plate electrodes and an electrical current is passed between the plates for about an hour in order for the molecules to electrophoretically transfer onto the membrane. Most netters prefer to use the standard commercial blotters such as the Panther [TM] HEP-1 from Owl Scientific, the Trans-Blot SD Transfer Cell from Bio-Rad, or the Model TE70 SemiPhor from Pharmacia/Hoefer. Although these units act very similarly, netters say that one advantage is for the system to have coated platinum plates instead of graphite plates because the platinum ones last much longer. A disadvantage of these is that they are not all created equally, with some of them having the anode and cathode reversed, making the setup process different for each unit. In addition, all of the units are very expensive. Okay...want to save money on a semi-dry blotting apparatus? How about making your own from old electric motor parts? Some more-daring netters have tried their hand at building homemade versions of semi-dry blotters from pre-fabricated graphite slabs. One such transfer unit was described by Ed Rybicki (ed@molbiol.uct.ac.za), who says that his lab uses graphite blocks that are normally installed as brushes on large electric motors cut to 1 cm x 20 cm x 15 cm. The blocks are converted into electrodes by inserting automobile battery terminals into holes drilled within the sides, and then gluing them into place with silicone cement. A picture of the setup is available from http://www.uct.ac.za/microbiology/westblot.gif and instructions for using this unit can be found at http://www.uct.ac.za/microbiology/western.htm Dr Rybicki says that the blocks made from machine parts last for years, and when the graphite begins to disintegrate, they simply buy more electrodes as replacements. He does warn that the same electrodes should be clearly marked and used as either cathode or anode every time or else they flood the system with colloidal carbon. Also, extreme care should be taken not to touch this device while it is operating because the experimental results can be quite shocking! He suggests rigging the unit in such a way as to prohibit its use without the lid in place. All this over toothpicks? ************************* The `net' controversy over toothpicks continues...Tom Cooper (tcooper@bcm.tmc.edu) pointed out that the main issue covered in his article cited in this column (TiBS 22, 68-69) is that much less Taq polymerase can be used for PCR colony screening if wooden toothpicks are avoided. He says that plastic picks are not that much more expensive if bought in bulk from Soodhalter Plastics, Inc., Los Angeles, CA 90021, USA, and that netters concerned with cost should reconsider the value of their precious enzyme. References ********** [1] Hartman, P. S. (1991) BioTechniques 11, 747-748 [2] Grundemann, D. and Schomig, E. (1996) BioTechniques 21, 898-903 [3] Takeuchi, M. and Fujisawa, H. (1995) Anal. Biochem. 224, 611-612 ******************************************************************************* 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/may97.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1997. Methods and reagents: Protecting Vector DNA from UV light. Trends in Biochemical Sciences 22(5):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 /--------------------------/* *******************************************************************************