Methods and reagents - Bummer buffers and lightning ligations Got a bummer buffer? Your sonicator giving you bad vibes? Got the Coomassie blues? Your restriction enzyme from NZ-mart, the basement-price NZyme company, just won't cut it anymore? I know just the place for you: a newsgroup available on the Internet called bionet.molbio.methds-reagnts. Scientists, researchers and graduate students of molecular biology and biochemistry from all over the world meet at their own convenience and on their own computers to discuss topics ranging from the most simplistic question of where to buy chemicals and media, to the highly technical and often elusive control of gene amplification by the polymerase chain reaction (PCR). Come on in, relax, and have a look around. Cafe Bionet is open to the public 24 hours a day and it's free. Many scientists unfamiliar with the `net' are often astounded by the speed and ease of connectivity when reaching out for help with laboratory practices for which only a few people have expertise. All you have to do is post a message on the newsgroup electronic bulletin board outlining the problems you are having with, for example, your Southern or western blots, and within hours and sometimes even minutes, you can have the answer to your question. For example, I recently needed help in selecting the best buffers, membranes and electrophoresis conditions for electroblotting proteins onto polyvinylidine difluoride (PVDF) membranes. I posted a list of questions that had arisen during our initial attempts to electrophorese and transfer proteins onto a support matrix using this technique. Within hours I had e-mail streaming in from all parts of the world including Canada, Australia, South Africa, and Japan, offering free advice and experiences related to my designed experiments. Some came from old friends on the net and some came from people I had never even met before, but who were willing to spare me from the agony of learning by trial and error. From this, I could select the most sensitive and reliable membrane for my needs based on what other researchers had told me, without contacting any (possibly biased) sales representatives or having done a single experiment. Undoubtedly, the best tool I have found for my research is the Internet. Last year I volunteered to keep track of questions that frequently arise in the methods newsgroup. I have now been asked to keep a watchful eye out for interesting ideas and discussions there, and then write this column describing some of these. This will by no means be a comprehensive review of all the articles posted, which would be impossible given the breadth of discussions. It will, however, focus on just a few of the interesting threads discussed each month, most of which never appear in published form. So here it is: the first installment of a series of monthly articles designed to highlight some of the more interesting and current topics within the methods and reagents newsgroup known as bionet.molbio.methds-reagnts (Box 1). Ultrasonic DNA ligations ************************ Everyone would like to reduce the time needed for a ligation reaction, right? Well, how about using ultrasonic waves to jiggle the DNA molecules together? Sounds absurd? Think again! Researchers at the University of California, Santa Barbara have recently published an article [1] describing conditions which will yield transformation efficiencies `as great or greater than' the usual overnight ligations in just 5-15 minutes. This is accomplished by placing the ligation mixture in an ultrasonic cleaning bath. It was reported that performing plasmid blunt-end ligations at 16 degrees C for 15 minutes with ultrasound mimicked the efficiency of that performed at 16 degrees C for 16 hours without ultrasound. Many of the `netters' (people who gather on the net) were quite skeptical of this technique, claiming that they had tried this using an ultrasonic cleaning bath other than the Fisher Model FS-14 used in the original paper and that it did not work. Some were critical of the lack of technical conditions described. For example, it was not mentioned which setting was used or if the frequency could be adjusted on the unit. The specifications of the cleaning bath also were not mentioned. One worker however, Ari-Matti Saren (saren@operoni.helsinki.fi) wrote that the Branson 2200 ultrasonic cleaning bath (Branson Ultrasonics Corporation, Danbury, CT, USA) with high-frequency output power of 60 W and operating frequency of 47 kHz (+/- 6%) did work for their ligations. A phone call to Branson revealed that there are two versions of the 2200 model available, one heated with 234 W output and one unheated with 125 W output, both of 47 kHz frequency. Disappointingly, the exact conditions and results of the ligation experiments were not discussed. Clearly, a more robust study of various conditions favoring this type of ligation is needed to allow others to reproduce the high transformation efficiency reported. Optimization of this technique could save researchers many hours of waiting for their ligation reactions. Contamination of thermocycling machines by 35-S *********************************************** A discussion arose regarding contamination of PCR thermocycler machine heating/cooling blocks with the radioisotope 35-S during the course of cycle sequencing with 35-S labeled deoxynucleotides. The apparent source of the contamination observed by some workers was thought not to be from aerosols resulting from condensation of liquid from the microtubes during the thermocycling, but from an unidentified sulphur-containing breakdown product of the labeled nucleotides seeping directly through the thin-walled style of PCR tube or microtiter plates. This occurred on a regular basis since the block was contaminated after every PCR reaction no matter how much care was taken. Mike Finney (finney@Frodo.MGH.Harvard.EDU) suggested that the chemical generated during the breakdown may be radioactive hydrogen sulfide, although there is little evidence to support this except that the contamination is rarely observed in thick-walled 0.5 ml tubes. Contamination problems may also be complicated by the use of cap strips for use with different batches of PCR tubes that may vary in mouth diameter, leading to escape through the lip contacts. The contamination observed by many people on the net seems to be common and could not be attributed to any one brand of radioisotope or the age of the material used. The `seepage' problem, if that is what it is, appears to be almost completely avoided by using thick-walled tubes and 33-P labeled nucleotides. =============================================================================== Box 1. How to join the bionet.molbio.methds-reagnts newsgroup ************************************************************* Getting started with bionet is easy. Trying to limit yourself from spending many, many hours on the net is much more difficult. There are basically two ways to join in. First, you may have access to a newsreader via the computer facility in your own University Department of Computer Science. Ask your local computer systems administrator if you can get access to a newsreader at your school or research center. If you have access, all you have to do is run the reading and posting program within your own computer system and follow the prompts to subscribe to bionet.molbio.methds-reagnts. If your local computer administration does not yet have this capability, it is possible to obtain the required news software elsewhere. Information about how to get news software can be requested by e-mail to biosci@net.bio.net. The second way to get involved is by reading and/or posting within the newsgroup via e-mail. The main drawback to e-mail access as opposed to using news software is that messages will be mixed in with your personal e-mail and sorted only by the time of posting. News software is much smarter than mail and will actually group messages of similar subject together under each newsgroup so that discussions can be followed more easily. Your personal mail is not cluttered when you use news software. However, for many people e-mail access is the only means available for participation. If the only way you can get newsgroups is by e-mail, this is how: Subscribing/Unsubscribing by e-mail *********************************** First, you must subscribe to a newsgroup. To subscribe, select a newsgroup from the list provided by BIOSCI. Send an e-mail message to biosci@net.bio.net if you are within the Americas or Pacific Rim, or to biosci@daresbury.ac.uk if you are in Europe, Africa, or Central Asia. In the body of the message, request that you be added to the subscription list for that newsgroup. To unsubscribe, simply do the same, requesting removal from a newsgroup. Posting by e-mail ***************** To post, select the newsgroup for which you would like to post from the list provided by BIOSCI and send your message as if it were regular e-mail. The latest list of mailing addresses is found in the BIOSCI information sheet for your region. The BIOSCI information sheet can be requested from biosci@net.bio.net if you are within the Americas or Pacific Rim, or from biosci@daresbury.ac.uk if you are in Europe, Africa, or Central Asia. For example, to post an article in bionet.molbio.methds-reagnts, send your message to one of the following two addresses depending upon your location: Address.......................................Serving methods@net.bio.net..................The Americas and Pacific Rim methods@daresbury.ac.uk..........Europe, Africa, and Central Asia Do not send subscription requests to these addresses, or you will have posted it to the newsgroup on thousands of computers. New users of BIOSCI/bionet should subscribe to bionet.announce and bionet.general to keep up with current events on bionet. They should also read the Frequently Asked Question or "FAQ" sheet for BIOSCI. FAQs exist for many newsgroups and are a good source of information. The BIOSCI FAQ provides details on how to participate in these forums and is available by anonymous file transfer protocol (FTP) from net.bio.net [134.172.2.69] in pub/BIOSCI/biosci.FAQ. It may also be requested by sending e-mail to biosci@net.bio.net. The BIOSCI FAQ is also posted on the first of each month to the newsgroup bionet.announce immediately following the posting of the BIOSCI information sheet. Before posting any articles, it is generally a good idea to watch the discussion for a while to get a feeling for how people interact by computer. This is called `lurking', and you will be a `lurker' if you read but don't post. Keep in mind that if you post, your typed message may be read by thousands of people around the world. Some questions have been asked and answered several times before or discussed ad nauseam. To avoid many redundant questions, a FAQ list is usually available. Before posting, it is considered good `netiquette' for the poster to check the FAQ list for an answer to his/her question first so as not to bore those who participate in the discussions regularly. The FAQ list for bionet.molbio.methds-reagnts is available by anonymous FTP from ftp.ncifcrf.gov [note: the published version of this has the old address of ncifcrf.gov which is no longer valid] as the file pub/methods/FAQlist. It is also available by anonymous FTP from net.bio.net as the file pub/BIOSCI/METHDS-REAGNTS/METHODS.FAQ. To get a copy of the FAQ list for bionet.molbio.methds-reagnts by e-mail, you may send a message to the author at pnh@ncifcrf.gov. FTP archives of all the BIOSCI/bionet messages are available at net.bio.net [134.172.2.69] in /pub/BIOSCI and can also be retrieved by gopher. Contact biosci@net.bio.net for further help or comments on the archives. =============================================================================== Reference [1] Cooper, J. B., Halverson, D. L., and Coldren, C. (1993) Nucleic Acids Res. 21, 1681 ******************************************************************************* 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/nov93.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1993. Methods and reagents - Bummer buffers and lightning ligations. Trends in Biochemical Sciences 18(11):446-448. ******************************************************************************* * 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 /--------------------------/* *******************************************************************************