tag:blogger.com,1999:blog-68111369968574343062024-03-08T08:46:09.506+01:00LabTutorials in BiologyThis is a blog for basic techniques in molecular biology for beginners.Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.comBlogger35125tag:blogger.com,1999:blog-6811136996857434306.post-58023122865529045942011-03-26T13:31:00.000+01:002011-03-26T13:31:06.399+01:00Havasi - Drum & Piano<iframe width="480" height="295" src="http://www.youtube.com/embed/F_dHSEoMPFQ?fs=1" frameborder="0" allowFullScreen=""></iframe>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-42260604806239706852010-11-08T19:19:00.000+01:002010-11-08T19:19:00.859+01:00The Gibson Assembly Song<object style="background-image:url(http://i4.ytimg.com/vi/WCWjJFU1be8/hqdefault.jpg)" width="480" height="295"><param name="movie" value="http://www.youtube.com/v/WCWjJFU1be8?fs=1&hl=hu_HU"><param name="allowFullScreen" value="true"><param name="allowscriptaccess" value="always"><embed src="http://www.youtube.com/v/WCWjJFU1be8?fs=1&hl=hu_HU" width="480" height="295" allowScriptAccess="never" allowFullScreen="true" wmode="transparent" type="application/x-shockwave-flash"></embed></object>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-43199815071644384002010-08-30T16:29:00.001+02:002010-08-30T16:34:32.102+02:00Love and Other DrugsA good demonstration about the importance of pharmacology :)))<br />
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<object width="640" height="385"><param name="movie" value="http://www.youtube.com/v/TO0X4DQZKqk?fs=1&hl=hu_HU"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/TO0X4DQZKqk?fs=1&hl=hu_HU" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="640" height="385"></embed></object>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-27169960817090372762010-08-28T12:43:00.000+02:002010-08-28T12:43:14.241+02:00Liquid Nitrogen Experiments: The Rubber Stopper<object style="background-image:url(http://i2.ytimg.com/vi/yg45ILXZ26w/hqdefault.jpg)" width="480" height="295"><param name="movie" value="http://www.youtube.com/v/yg45ILXZ26w?fs=1&hl=en_US"><param name="allowFullScreen" value="true"><param name="allowscriptaccess" value="always"><embed src="http://www.youtube.com/v/yg45ILXZ26w?fs=1&hl=en_US" width="480" height="295" allowScriptAccess="never" allowFullScreen="true" wmode="transparent" type="application/x-shockwave-flash"></embed></object>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-51096083952162878212010-08-28T12:41:00.000+02:002010-08-28T12:41:15.618+02:00Liquid Nitrogen Experiments: Insulators<object style="background-image:url(http://i3.ytimg.com/vi/6WnGHhMKUuk/hqdefault.jpg)" width="480" height="295"><param name="movie" value="http://www.youtube.com/v/6WnGHhMKUuk?fs=1&hl=en_US"><param name="allowFullScreen" value="true"><param name="allowscriptaccess" value="always"><embed src="http://www.youtube.com/v/6WnGHhMKUuk?fs=1&hl=en_US" width="480" height="295" allowScriptAccess="never" allowFullScreen="true" wmode="transparent" type="application/x-shockwave-flash"></embed></object>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com1tag:blogger.com,1999:blog-6811136996857434306.post-45657590753109747342010-08-28T12:38:00.000+02:002010-08-28T12:38:43.529+02:00Dry Ice vs. Liquid Nitrogen<object style="background-image:url(http://i4.ytimg.com/vi/ctPbhKldOgA/hqdefault.jpg)" width="480" height="295"><param name="movie" value="http://www.youtube.com/v/ctPbhKldOgA?fs=1&hl=en_US"><param name="allowFullScreen" value="true"><param name="allowscriptaccess" value="always"><embed src="http://www.youtube.com/v/ctPbhKldOgA?fs=1&hl=en_US" width="480" height="295" allowScriptAccess="never" allowFullScreen="true" wmode="transparent" type="application/x-shockwave-flash"></embed></object>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-19850917081289736932010-08-28T12:36:00.000+02:002010-08-28T12:36:15.209+02:00Liquid Nitrogen Ice Cream - Short Version<object style="background-image:url(http://i2.ytimg.com/vi/ePYuJIWF1h8/hqdefault.jpg)" width="425" height="344"><param name="movie" value="http://www.youtube.com/v/ePYuJIWF1h8?fs=1&hl=en_US"><param name="allowFullScreen" value="true"><param name="allowscriptaccess" value="always"><embed src="http://www.youtube.com/v/ePYuJIWF1h8?fs=1&hl=en_US" width="425" height="344" allowScriptAccess="never" allowFullScreen="true" wmode="transparent" type="application/x-shockwave-flash"></embed></object>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-11835309335649698512010-08-28T12:34:00.000+02:002010-08-28T12:34:52.285+02:00Liquid Nitrogen Experiments: The Carnation<object style="background-image:url(http://i4.ytimg.com/vi/Gz3lrLoW0og/hqdefault.jpg)" width="480" height="295"><param name="movie" value="http://www.youtube.com/v/Gz3lrLoW0og?fs=1&hl=en_US"><param name="allowFullScreen" value="true"><param name="allowscriptaccess" value="always"><embed src="http://www.youtube.com/v/Gz3lrLoW0og?fs=1&hl=en_US" width="480" height="295" allowScriptAccess="never" allowFullScreen="true" wmode="transparent" type="application/x-shockwave-flash"></embed></object>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-38296931231761443122010-08-28T12:33:00.000+02:002010-08-28T12:33:15.772+02:00Liquid Nitrogen vs. Liquid Oxygen: Fire<object style="background-image:url(http://i2.ytimg.com/vi/iKIi8KLLvbA/hqdefault.jpg)" width="425" height="344"><param name="movie" value="http://www.youtube.com/v/iKIi8KLLvbA?fs=1&hl=en_US"><param name="allowFullScreen" value="true"><param name="allowscriptaccess" value="always"><embed src="http://www.youtube.com/v/iKIi8KLLvbA?fs=1&hl=en_US" width="425" height="344" allowScriptAccess="never" allowFullScreen="true" wmode="transparent" type="application/x-shockwave-flash"></embed></object>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-68586376213954257652010-08-28T12:26:00.000+02:002010-08-28T12:26:39.588+02:00Liquid Nitrogen Demonstration<object style="background-image:url(http://i2.ytimg.com/vi/5j02WUaIF1E/hqdefault.jpg)" width="425" height="344"><param name="movie" value="http://www.youtube.com/v/5j02WUaIF1E?fs=1&hl=en_US"><param name="allowFullScreen" value="true"><param name="allowscriptaccess" value="always"><embed src="http://www.youtube.com/v/5j02WUaIF1E?fs=1&hl=en_US" width="425" height="344" allowScriptAccess="never" allowFullScreen="true" wmode="transparent" type="application/x-shockwave-flash"></embed></object>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-18553507959693025472010-08-28T12:13:00.000+02:002010-08-28T12:13:25.864+02:00Cooking with Liquid Nitrogen - Ferran Adria and Harold McGee<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/t3VPeyYL-fI?fs=1&hl=en_US"><param name="allowFullScreen" value="true"><param name="allowscriptaccess" value="always"><embed src="http://www.youtube.com/v/t3VPeyYL-fI?fs=1&hl=en_US" width="425" height="344" allowScriptAccess="never" allowFullScreen="true" wmode="transparent" type="application/x-shockwave-flash"></embed></object>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-45397332195860474032010-08-24T15:39:00.000+02:002010-08-24T15:39:50.506+02:00A men for Science Education after receiveng his Nobel PrizeCarl Wieman, is a Nobel Laureate in Physics. He gave in the last years a series of lectures about the scientific assesment of teaching science. <br />
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On of these lectures about how to teach science given at Harvard can be seen <a href="http://t7.hu/f01">here</a>.<br />
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The conclusions are refreshing: there is a way of teaching science outside the laboratories too. <br />
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One of the tools he is suggesting is modelling of real experiments. He developedd and made freely available a set of basic experiments.<br />
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You can have a look to these and use them at the website of the Colorado University <a href="http://phet.colorado.edu/en/simulations/category/biology">here</a>.<br />
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In his lectures he is refering to a book abour expertieze end experts, The Cambridge Handbook of Expertise and Expert Performance. Seems to be a relevant book int this topic:<br />
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<iframe src="http://rcm.amazon.com/e/cm?t=labtuto-20&o=1&p=8&l=bpl&asins=0521600812&fc1=000000&IS2=1<1=_blank&m=amazon&lc1=0000FF&bc1=000000&bg1=FFFFFF&f=ifr" style="align:left;padding-top:5px;width:131px;height:245px;padding-right:10px;"align="left" scrolling="no" marginwidth="0" marginheight="0" frameborder="0"></iframe>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-71752017198717909782010-06-30T21:55:00.001+02:002010-07-24T16:59:33.138+02:00I moved!!!Dear colleagues, <br />
<br />
I moved from wordpress to Blogger using this excellent tool:<br />
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<a href="http://www.ampercent.com/wordpress-to-blogger-migration/4882/">http://www.ampercent.com/wordpress-to-blogger-migration/4882/</a><br />
<br />
Best,<br />
<br />
Balint.Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-75948958945614503062010-06-30T20:09:00.003+02:002010-07-25T10:55:05.907+02:00Sizes in BiologyDear colleagues,<br />
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An excellent tool is available to get a feeling about molecular sizes in Biology.<br />
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The tool is available <a href="http://learn.genetics.utah.edu/content/begin/cells/scale/" target="_blank" title="Cell sizes">here</a> and was developped by "Learn Genetics" program from the University of Utah.<br />
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You can have a short movie on it below, but please check the original one from <a href="http://learn.genetics.utah.edu/content/begin/cells/scale/" target="_blank" title="Cell sizes">here</a>!!!<br />
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<object height="385" width="480"><param name="movie" value="http://www.youtube.com/v/l7kZjdeo0Cs&hl=hu_HU&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/l7kZjdeo0Cs&hl=hu_HU&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="480" height="385"></embed></object><br />
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You can also have a deep immersion in the issue here:<br />
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<div style="text-align: center;"><br />
</div><div style="text-align: center;"><iframe align="left" frameborder="0" marginheight="0" marginwidth="0" scrolling="no" src="http://rcm.amazon.com/e/cm?t=labtuto-20&o=1&p=8&l=bpl&asins=0691128502&fc1=000000&IS2=1&lt1=_blank&m=amazon&lc1=0000FF&bc1=000000&bg1=FFFFFF&f=ifr" style="height: 245px; padding-right: 10px; padding-top: 5px; width: 131px;"></iframe></div>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-85925386394541906142010-06-08T23:19:00.001+02:002010-07-25T11:24:38.368+02:00Designing a gene or "Gene design"On this tutorial you can see an easy way to design oligos for gene synthesis, or in other words Gene design.<br />
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The description is based on the <a href="http://2009.igem.org/Instructional_Videos">Instructional Videos</a> from the iGEM website!<br />
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Gene design in less than 10 minutes!<br />
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Good luck!!!<br />
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<object width="480" height="385"><param name="movie" value="http://www.youtube.com/v/zaXCbX2NmNU&hl=hu_HU&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/zaXCbX2NmNU&hl=hu_HU&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="480" height="385"></embed></object><br />
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Some usefull tools:<br />
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1. <a href="http://baderlab.bme.jhu.edu/gd/">Gene design web page</a><br />
2. <a href="https://www.dna20.com/tools.php">Tools at DNA20</a><br />
3. <a href="http://helixweb.nih.gov/dnaworks/">Tools at DNA Works</a><br />
4. <a href="http://www.bioinformatics.org/sms/rev_comp.html">A Revese-Complement Tool</a><br />
5. <a href="https://www.dna20.com/genedesigner2/">Gene designer</a> a comprehensive tool to artificial gene design from DNA20 described <a href="http://en.wikipedia.org/wiki/Gene_Designer">here.</a><br />
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<br />
If you would like to read about the application of design principles in Drug Discovery you should read this book:<br />
<iframe align="left" frameborder="0" marginheight="0" marginwidth="0" scrolling="no" src="http://rcm.amazon.com/e/cm?t=labtuto-20&o=1&p=8&l=bpl&asins=0470412895&fc1=000000&IS2=1&lt1=_blank&m=amazon&lc1=0000FF&bc1=000000&bg1=FFFFFF&f=ifr" style="height: 245px; padding-right: 10px; padding-top: 5px; width: 131px;"></iframe> Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com1tag:blogger.com,1999:blog-6811136996857434306.post-41097841704396670952010-05-16T14:29:00.000+02:002010-07-24T16:55:07.209+02:00IDEA Generation and Brain stormingOne of the first step in generating a project is to generate ideas. One of the best known technique for idea generation is <a href="http://en.wikipedia.org/wiki/Brainstorming">Brainstorming</a>.<br/><br/>Below you can see excerpts from the very well writen and detailed description of the Wikipedia:<br/><h3>Basic rules:</h3><br/>There are four basic rules in brainstorming. These are intended to reduce social inhibitions among group members, stimulate idea generation, and increase overall creativity of the group.<br/><ol><br/> <li><strong>Focus on quantity</strong>.</li><br/> <li><strong>Withhold criticism.</strong> In brainstorming, <a title="Criticism" href="http://en.wikipedia.org/wiki/Criticism">criticism</a> of ideas generated should be put 'on hold'. By suspending judgment, participants will feel free to generate unusual ideas.</li><br/> <li><strong>Welcome unusual ideas</strong>. They can be generated by looking from new perspectives and suspending assumptions. These new ways of thinking may provide better solutions.</li><br/> <li><strong>Combine and improve ideas</strong>: Good ideas may be combined to form a single better good idea, as suggested by the slogan "1+1=3". It is believed to stimulate the building of ideas by a process of <a title="Association (psychology)" href="http://en.wikipedia.org/wiki/Association_(psychology)">association</a>.</li><br/></ol><br/><h3> </h3><br/><h3>Method</h3><br/><h3> </h3><br/><h3>Set the problem</h3><br/>Before a brainstorming session, it is critical to define the problem.<br/><h3>Create a background memo</h3><br/>The background memo is the invitation and informational letter for the participants, containing the session name, problem, time, date, and place. The problem is described in the form of a question, and some example ideas are given. The memo is sent to the participants well in advance, so that they can think about the problem beforehand.<br/><h3>Select participants</h3><br/>1. The facilitator<br/><br/>2. Participants:<br/><ul><br/> <li>a. core members</li><br/> <li>b. guests from outside the project, with affinity to the problem.</li><br/></ul><br/>3. One idea collector who records the suggested ideas.<br/><h3> </h3><br/><h3>Create a list of lead questions</h3><br/>The facilitator should stimulate creativity by suggesting a lead question to answer, such as <em>Can we combine these ideas?</em> or <em>How about looking from another perspective?</em>. It is best to prepare a list of such leads before the session begins.<br/><br/>Another alternative method that could be used even on-line is the one described as:<br/><br/>"Group passing technique<br/><br/>Each person in a circular group writes down one idea, and then passes the piece of paper to the next person in a clockwise direction, who adds some thoughts. This continues until everybody gets his or her original piece of paper back. By this time, it is likely that the group will have extensively elaborated on each idea.<br/><br/>The group may also create an "Idea Book" and post a distribution list or routing slip to the front of the book. On the first page is a description of the problem. The first person to receive the book lists his or her ideas and then routes the book to the next person on the distribution list. The second person can log new ideas or add to the ideas of the previous person. This continues until the distribution list is exhausted. A follow-up "read out" meeting is then held to discuss the ideas logged in the book. This technique takes longer, but it allows individuals time to think deeply about the problem."<br/><br/>I think an iGEM group could create these tools for the group on the google group page which the members can access at the group site.<br/><br/>Another very good web site in the topic is <a title="JBP" href="http://www.jpb.com/index.php" target="_blank">JBP</a>.<br/><br/>Have fun!Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com1tag:blogger.com,1999:blog-6811136996857434306.post-80217760384579546642010-05-16T13:06:00.000+02:002010-07-24T16:55:07.193+02:00What is a COMMITMENT?<div>Dear Collegues,</div><br/><div> </div><br/><div>Please find below some words of <a title="http://www.yehudaberg.com/" href="http://www.yehudaberg.com/" target="_blank">Yehuda Berg </a>about what a Commitment means.</div><br/><div> </div><br/><div>"...a commitment is ongoing. It needs to be made and remade every day.</div><br/><div>If you decide to run a marathon, you aren’t just choosing to show up on the day of the event.</div><br/><div>You’re choosing a path, and that means conditioning every day. If you skip a week of training, you might not make it to the marathon.</div><br/><div>Being committed to a spiritual path means sometimes we’ll be alone. We might feel like a black sheep. Other times, people might want us to fail. Or at least when we succeed, their insecurities will be awakened and they won’t be happy about our success.</div><br/>And one thing’s for sure: we will be tested. Not on our decision, but on our commitment."<br/><div><br/><div>Starting a project, any project, needs a commitment.</div><br/><div>A succesfull iGEM teem needs a written commitment as described in the <a href="http://www.bio.davidson.edu/projects/gcat/Synthetic/Building_a_Team.pdf">paper of Wayne Materi</a>. We commit ourselfs to <span style="font-family:TimesNewRomanPSMT;">work hard, teach/learn lots and have fun.</span></div><br/></div><br/><div>Worth thinking about Yehuda's thoughts in general.</div><br/><div> </div><br/><div>Best,</div><br/><div> Balint.</div>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-6891086154567358312010-05-10T18:58:00.000+02:002010-07-24T16:55:07.155+02:00Prezi - a new tool for making better prezentationsOne of the most interesting development in the field of prezentation tools is <a href="http://prezi.com/learn/">Prezi</a>.<br/><br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/OMEUNahHNXc&hl=hu_HU&fs=1&"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/OMEUNahHNXc&hl=hu_HU&fs=1&;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>Beside using it for presentations, you can use it for Mind Mapping too, like here:<br/><br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/2UQh7UeTvek&hl=hu_HU&fs=1&"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/2UQh7UeTvek&hl=hu_HU&fs=1&;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>And here you can see some tricks for masters:<br/><br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/oIJ2cH9EyhA&hl=hu_HU&fs=1&"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/oIJ2cH9EyhA&hl=hu_HU&fs=1&;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>You can use it for free, and if you are using it for educational purposes, there is an edu version for it, with lot of cool features.<br/><br/>So, why not using a better tool for the same job?Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com2tag:blogger.com,1999:blog-6811136996857434306.post-25847859918596275152010-05-09T21:41:00.000+02:002010-07-24T16:55:07.071+02:00iGEM Hungary<strong>Dear Colleagues, </strong><br/><br/><strong>You might have heard about iGEM, the <a href="http://igem.org/">International Genetically Engineered Machine competition (iGEM)</a>. I personally became interested in this competition after Malcolm Campbell gave a lecture for us in December 2009. You can see his lectures including the one he gave at the University of Debrecen ( through video connection ) <a href="http://www.bio.davidson.edu/people/macampbell/macampbell.html#anchor66335659">here.</a> The group coordinated by Malcolm has an excellent Synthetic Bio summary <a title="Davidson SynBio" href="http://www.bio.davidson.edu/projects/gcat/Synthetic/synthetic.html" target="_blank">here</a>. </strong><br/><br/><strong>Synthetic Biology is about changing the design of biological entities (like vectors or small systems) into a more predictable, engineer type design.</strong><br/><br/><strong> </strong><br/><br/><strong>So we registered with a team Debrecen Hungary. I hope I can keep you updated with the developments.</strong><br/><br/><strong>The best short video about what synthetic biology is can be seen below.</strong><br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/XIuh7KDRzLk&hl=en_US&fs=1&"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/XIuh7KDRzLk&hl=en_US&fs=1&;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-68166201929979952522009-05-19T18:57:00.000+02:002010-07-24T16:55:07.054+02:00QPCR oligo designDesigning QPCR oligos might seem complicated but there are some rules and softwares that can make it easy. In our lab, majority of data points we generate are probably measured by QPCR, so I think it is worth to review an algorithm for desiging QPCR assays.<br/><br/>So, today I will describe the way I design QPCR oligos. You can have a basic intro in PCR <a title="PCR intro" href="http://labtutorials.org/2009/04/12/pcr-or-the-polymerase-chain-reaction/" target="_blank">here.</a><br/>More than a year ago I switched to the UPL system, the library of probes designed by Exiqon and now marketed by Roche. The concept is quite clear, the LNA modified oligonucleotides bind much stronger to the DNA template than the average oligonucleotides. By this we can decrease the lengths of them by keeping the Tm unchanged. The UPL library consists of 165 individual oligonucleotides that are in general nine basepair long and together cover the entire genome in respect of the coverage needed to design a QPCR oligo set for any gene. You can read more about the LNA nucleotides <a title="LNA Wiki" href="http://en.wikipedia.org/wiki/Locked_nucleic_acid" target="_blank">here</a> and <a title="Exiqon" href="http://www.exiqon.com/lna-technology" target="_blank">here</a>. A good website where you can calculate the Tm of the LNA oligos here: <a title="http://lna-tm.com/" href="http://lna-tm.com/" target="_blank">http://lna-tm.com/</a>. The UPL system is described <a title="UPL Roche" href="http://www.roche-applied-science.com/sis/rtpcr/upl/ezhome.html" target="_blank">here</a>.<br/><br/>The system allows the design of an oligo set for any DNA sequence in the <a title="UPL Design Center" href="http://www.universalprobelibrary.com/" target="_blank">UPL Design Centre</a>. You can access the Design Centre directly <a title="Assay design" href="https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000" target="_blank">here</a>.<br/><br/>[caption id="attachment_581" align="aligncenter" width="468" caption="UPL Assay Design Center"]<img class="size-full wp-image-581" title="UPL1" src="http://labtutorials.files.wordpress.com/2009/05/upl1.jpg" alt="UPL Assay Design Center" width="468" height="333" />[/caption]<br/><br/>Before we design an assay let us first look for the transcripts of a specific gene. It is very important to use annotated data, since in the annotated genomic data we have informations about possible SNP variations. This might be important, since the oligos and especially the probe should bind to an SNP free free region, because an SNP might disturb the binding of the probe to the template.<br/><br/>To make this data available we should use not the sequence but the transcript ID from the <a title="Ensembl" href="http://www.ensembl.org/index.html" target="_blank">Ensembl</a>.<br/><br/>[caption id="attachment_586" align="aligncenter" width="468" caption="Ensembl"]<img class="size-full wp-image-586" title="ensembl" src="http://labtutorials.files.wordpress.com/2009/05/ensembl.jpg" alt="Ensembl" width="468" height="443" />[/caption]<br/><br/>At Ensembl select your species of interest, e.g. mouse and write the name of your gene of interest in the <a title="Mouse search box" href="http://www.ensembl.org/Mus_musculus/Info/Index" target="_blank">search box</a>:<br/><br/><img class="aligncenter size-full wp-image-587" title="search box" src="http://labtutorials.files.wordpress.com/2009/05/search-box.jpg" alt="search box" width="468" height="222" /><br/><br/>If you write into the box a gene of interest (e.g. COUP-TF2) we will see the results as it is shown here:<br/><br/><img class="aligncenter size-full wp-image-589" title="couptf search" src="http://labtutorials.files.wordpress.com/2009/05/couptf-search.jpg" alt="couptf search" width="468" height="183" /><br/><br/>Here I have to click on the link of the gene and I will find the following screen: <img class="aligncenter size-full wp-image-591" title="transcript info" src="http://labtutorials.files.wordpress.com/2009/05/transcript-info.jpg" alt="transcript info" width="468" height="241" />The most important info we are looking for is in the table on the top of the page:<br/><br/><img class="aligncenter size-full wp-image-594" title="transcripts" src="http://labtutorials.files.wordpress.com/2009/05/transcripts.jpg" alt="transcripts" width="467" height="86" /><br/><br/>The two transcripts of the gene are:<br/>ENSMUST00000089565<br/>ENSMUST00000032768.<br/><br/>We will use these ID-s in the UPL Assay Design Centre. First select as organism the "Mouse", and write into the box the two Ensembl transcript ID-s selected by commas. <img class="aligncenter size-full wp-image-598" title="design" src="http://labtutorials.files.wordpress.com/2009/05/design.jpg" alt="design" width="468" height="394" />If you follow the steps the results will be like this:<br/><br/><img class="aligncenter size-full wp-image-599" title="res" src="http://labtutorials.files.wordpress.com/2009/05/res.jpg" alt="res" width="468" height="430" />Below this data you can see two links as it follows:<img class="aligncenter size-full wp-image-600" title="common assays" src="http://labtutorials.files.wordpress.com/2009/05/common-assays.jpg" alt="common assays" width="468" height="183" />To design an assay that would measure all transcripts select: "common assays".<br/><br/>The results will be given in a downloadable pdf report. Save this file and name is by the name of the gene you used as input.<br/><br/>The results in the pdf file look like this:<br/><br/><img class="aligncenter size-full wp-image-601" title="results" src="http://labtutorials.files.wordpress.com/2009/05/results.jpg" alt="results" width="468" height="322" /><br/><br/>You can see that the amplicon is 95 bp long, there is no SNP in the binding regions of the primers and probes and the probe is closer to one of the primers. There is an SNP in the gene that was avoided by the program. You can have an SNP even in the amplicon, unless is not in the binding site of the primers or the probe.<br/><br/>Before I order the oligos, I usually test them with e-PCR on the <a title="UCSC Genome Browser" href="http://genome.ucsc.edu/" target="_blank">UCSC Genome Browser</a>.<br/><br/>[caption id="attachment_608" align="aligncenter" width="468" caption="UCSC Genome Browser"]<img class="size-full wp-image-608" title="ucsc" src="http://labtutorials.files.wordpress.com/2009/05/ucsc.jpg" alt="UCSC Genome Browser" width="468" height="63" />[/caption]<br/><br/>Select the PCR view and paste the oligos into the given locations. Select the genome, the assembly and the target as "UCSC Genes"(If you used a genomic sequence for design use the target: "genome assembly").<br/><br/><img class="aligncenter size-full wp-image-609" title="in silico PCR" src="http://labtutorials.files.wordpress.com/2009/05/in-silico-pcr.jpg" alt="in silico PCR" width="467" height="70" />If you have a hit, click on the link provided and the results will be represented in the genome as seen here:<br/><br/><img class="aligncenter size-full wp-image-610" title="browser results" src="http://labtutorials.files.wordpress.com/2009/05/browser-results.jpg" alt="browser results" width="467" height="350" /><br/><br/>The oligos are intron spanning and in the right location. Order the oligos in an HPLC pufied form in the lowest available scale for the first try. Be aware that according to the experience of several groups, and my own experience too, only 2/3 of the UPL assays work without further optimization. This means, if you want to be sure from the first you better try two, three different assays for the same gene. The UPL Design Centre will generate several primer-probe sets and you can retrive these results too. Since the UPL library is given and one or two of the three ordered assays will work, it this worth trying three from the beginning!<br/><br/>In general the rules for a good QPCR assay:<br/><br/>1. The amplicon should be as short as possible (60-70 bp is ideal, but should be shorter than 100bp).<br/><br/>2. The Tm of the oligos should be around 60C, while the Tm of the probe 10C higher.<br/><br/>3. The distance between the the oligo and the probe should be as small as possible for a better exonuclease activity of the Taq polymerase.<br/><br/>4. The GC content of the two oligos should be as close as possible.<br/><br/>5. The number of GC bases in the last five nucleotides on the 3' ends of the two primers should be identical (if possible).<br/><br/>6. Select for oligo sets with week internal bonds (less than four H bonds in the same conformation).<br/><br/>7. Avoid primer dimers that could produce artefacts due to the 3' elongation of one of the primers. The same for internal conformations. See below:<img class="aligncenter size-full wp-image-618" title="conformations" src="http://labtutorials.files.wordpress.com/2009/05/conformations1.jpg" alt="conformations" width="468" height="190" /><br/><br/>8. Verify the oligos with e-PCR on the UCSC Genome Browser. The test should give one single hit!<br/><br/>9. If possible use annotated sequences to avoid the SNP effect.<br/><br/>10. If you are looking for genes (cDNA measurement) use exons that are common for all transcripts variants (or use the "batch assay-common assays" in the UPL Assay Design Centre)<br/><br/>Good luck!Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-75768666540533733032009-04-30T12:02:00.000+02:002010-07-24T16:55:07.002+02:00What you always wanted to know about virusesConsidering that swine flu is here everywhere and many became interested in viruses, I compiled here a few videos that might give you an update about viruses, virology, vaccine production and how a vaccine acts. Feedback and questions are welcome at<br/><br/>balintblaszlo(at)labtutorials(dot)org<br/><br/>Regards,<br/><br/>Balint.<br/><br/>Where are the swine flu cases?<br/><br/><a title="Healthmap" href="http://www.healthmap.org/swineflu" target="_self">See actual status here</a><br/><br/>or check it in GoogleMaps here:<br/><br/>[googlemaps http://maps.google.com/maps/ms?ie=UTF8&hl=en&t=p&msa=0&msid=106484775090296685271.0004681a37b713f6b5950&ll=32.639375,-110.390625&spn=15.738151,25.488281&output=embed&w=425&h=350]<br/>An overview of the status by 20090430:<br/><br/><img class="aligncenter size-full wp-image-569" title="healthmap200904301" src="http://labtutorials.files.wordpress.com/2009/04/healthmap200904301.jpg" alt="healthmap200904301" width="468" height="295" /><br/><br/>How does influenza spreads and what happens with it in our body?<br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/kgUEswnQM3M&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/kgUEswnQM3M&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>The life of a virus<br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/A409yO-G1Mk&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/A409yO-G1Mk&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>The structure of viruses<br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/7iVm1uEIyP0&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/7iVm1uEIyP0&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/><a title="vaccine production" href="http://www.ehponline.org/members/2006/114-2/innovations.html" target="_blank">Vaccine production</a>(description and images).<br/><br/>Vaccine production at <a title="Sanofi virus production" href="http://www.sanofipasteur.com/sanofi-pasteur2/front/index.jsp?siteCode=SP_CORP&codeRubrique=64" target="_blank">Sanofi (video). </a><br/><br/>How does the vaccine act?<br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/3ykHoIKAtQI&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/3ykHoIKAtQI&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>Injection of the vaccine<br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/cv48WxiQ6Xw&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/cv48WxiQ6Xw&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>More about pandemics, and vaccination <a title="vaccine development" href="http://labtutorials.org/2009/04/29/bird-flu-solved-swine-flu-arrived/" target="_self">here</a>.<br/><br/>How microarrays can be used for rapid characterization of viruses: <a title="microarray and virology" href="http://labtutorials.org/2009/03/30/hunting-viruses/" target="_self">here</a>.<br/><br/>Stay tuned!Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com2tag:blogger.com,1999:blog-6811136996857434306.post-46925587628988919252009-04-29T18:12:00.000+02:002010-07-24T16:55:06.958+02:00Bird flu solved, swine flu arrived!<h3 class="UIIntentionalStory_Message">The bird flu solved by vaccines, now comes the swine flu.</h3><br/>Pandemics shaped the human population in past and probably in future. One of the most destructive influenza pandemics in the last century was the Spanish Flu which caused the death of 25-40 million people. More info about the pandemics <a title="Pandemics" href="http://www.omninvest.hu/influenza/english/jarvanyok.html" target="_blank">here</a>. The very last thread to cause a pandemic is the virus called A/H1N1, or swine flu, or Mexican flu.<br/><br/>Rapid diagnostic tests will be probably be <a title="PCR intro" href="http://labtutorials.org/2009/04/12/pcr-or-the-polymerase-chain-reaction" target="_blank">PCR</a> based. A very interesting microarray based virus characterization tool is described <a title="Virus hunting with microarrays" href="http://labtutorials.org/2009/03/30/hunting-viruses/" target="_blank">here</a>.<br/><br/>Influenza vaccines are produced by quite a lot of manufacturers in the world. You can have a list of the manufacturers <a title="Flu vaccine manufacturers" href="http://www.omninvest.hu/influenza/english/gyartas.html" target="_blank">here</a>.<br/><br/>A small Hungarian company is a word leader in vaccine development. <a title="Omninvest" href="http://www.omninvest.hu/influenza/english/index.html" target="_blank">Omninvest</a> was one of the first who produced the anti H5N1 vaccine. It is good to draw the conclusions of the Bird Flu vaccine development, a detailed overview can be found <a title="H5N1 vaccine" href="http://www.omninvest.hu/influenza/english/h5n1.html" target="_blank">here</a>.<br/><br/>Today Omninvest announced that they made all the preparatory steps needed to start to develop a Swine Flu vaccine. Once the sample virus arrives they can start the development of inactivated viruses that can be used for the development of the Swine flu vaccine. While US based CDC researcher Ruben Donis announced that the <a href="http://www.google.com/hostednews/ap/article/ALeqM5gcx9bjqSn_mHLMw5rb3eoY32TZdQD97RKIJ00" target="_blank">"reference strain" would be available </a>at the beginning of May, the Hungarian news agency MTI released the information that samples that could be used for further development migh <a title="Swine Flu sample" href="http://english.mti.hu/default.asp?menu=1&theme=2&cat=25&newsid=261275" target="_blank">arrive in days</a>.<br/><br/>For more details about the life of a virus and vaccination check <a title="Influenza" href="http://labtutorials.org/2009/04/30/about-viruses/" target="_self">this</a>.Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com2tag:blogger.com,1999:blog-6811136996857434306.post-86649495232041480252009-04-29T12:08:00.000+02:002010-07-24T16:55:08.827+02:00Dear All,<br/><br/>In this changing world we all feel the pressure to find novel ways to increase our efficiency and impact. Recently I just came in contact with Howard Wolinsky a freelancer scientific and medical journalist. The way how Howard changed the pace of his work seems to reflect the "flat world" paradigm. We will discuss about journalism, being a freelancer, scientific journalism, and the impact of web based networking via Facebook, LinkedIn, Twitter and so on.<br/><br/>So let us have a short interview with Howard Wolinsky:<br/><br/>----------------------------------------------------------------------------------------<br/><br/>LTB: After working so many years as an employee, you decided to become a freelancer. What was your strongest motivation to make this step?<br/><br/>HW: I started out as a newspaper reporter in a small town, Kankakee, Ill., USA, 1970, covering mental health (there were two large mental institutions in town) and local government.<br/><br/>But I had my eyes on bigger things. I wanted to be a health/medical reporter for a major daily.<br/><br/>My colleagues often described me as "entrepreneurial." That meant, I freelanced and they didn't for the most part.<br/><br/>I started to freelance articles to the Associated Press and United Press International. I did a story for the Chicago Daily News--an historic newspaper where Carl Sandburg, the poet, and Ben Hecht, the playwright, who authored "Front Page," had worked--on a mental health topic. I was offered another assignment.<br/><br/>But the Kankakee Journal nearly fired me for writing for a "competitor."<br/><br/>But I kept freelancing throughout the years. Some years, as I moved on to other papers, including St. Petersburg (Fla.) Times, Florida Today (then in Cocoa, Fla.) and Chicago Sun-Times—my freelance wages matched or exceeded my base pay.<br/><br/>I considered leaving to be a full-time freelancer a couple times. But one of the problems in the US is health insurance. One time, on the verge of freelancing full-time, I contracted pneumonia and was out for a couple weeks. My pay at the Sun-Times continued. But had I only had freelance, I would have had financial worries. No work, no pay as a freelance.<br/><br/>In the intervening years, my wife returned to work and could offer us insurance coverage.<br/><br/>As the economics of the newspaper business deteriorated in general and at the Chicago Sun-Times in particular, I had an opportunity to take a buy-out after nearly 27 years on the job as a health and tech reporter. As I described in an article in GaperBlock.com, I had a flash of insight on how I could take a year's pay as "seed money" to start my own freelance business.<br/><br/>I left on a Friday in January 2008. The next Monday I had my first assignment.<br/><br/>But in fact, I unconsciously been preparing for this move since 1970.<br/><br/>A friend once advised me that I shouldn't just have one job but multiple ones. That's what I did last year. In addition to freelance writing, I diversified. I went in new directions. I networked.<br/><br/>I became the US blogger for Skype. I taught at Medill Graduate School of Journalism at Northwestern University. I wrote for university/medical school magazines, such as Cleveland Clinic, Northwestern Memorial Hospital, Roswell Park Cancer Center, Howard Hughes Medical Institution, University of Illinois at Chicago. I wrote for BusinessWeek, more general, non-science stories. (Check out my story in BW on David Axelrod, President Obama's advisor.) I was invited to contribute to Huffington Post, which doesn't pay (yet) but gave me a chance to stretch and write in new areas. I wrote for my former competitor, Chicago Tribune. I wrote some articles for Venture Capital Journal. I did some consulting. I started doing more for my friends at EMBO Reports. I did a number of stories for WebVet.com. On and on.<br/><br/>And as busy as I was, I spent two months traveling. My wife and I celebrated her birthday and the Inca New Year at a yoga retreat in Peru (we were remarried by a shaman at Machu Picchu). The BBC paid my way to UK to appear on the Coast program. We went to Paris on the train for a day. Took a couple trips to Northern California and one to Florida.<br/><br/>LTB: You are doing plenty of different type of activities: you write books, you are a blogger for Skype, US correspondent for EMBO Reports, you are teaching and this is only the tip of the iceberg. How do you manage to keep your focus? What is your secret of productivity?<br/><br/>HW: Some days, I do feel that I have taken on too much. I am a fast writer, which helps. I also have turned away work if I didn't find it interesting, but I always tried to steer the work to a friend. It's important to network. (One of the things I did everyday after I left my full-time job was spend an hour a day building my network at LinkedIn, Facebook and now Twitter. Jobs have come my way because people have found me in LinkedIn and Facebook--even though I can be readily found through a Google search.)<br/><br/>LTB: You are located in Chicago but a substantial part of your activity is targeting a much wider audience. Do you see a trend in this? Is the world really becoming flat? From practical point of view do you think people need a strong local connection network and mix this with a web based business or you see that targeting people trough the web could be enough in itself?<br/><br/>HW: I always told my kids to think globally because that's where the work will be. I also told them to become fluent in another language. (I am working at that now. I am taking Spanish via Skype with a tutor in Guatemala.)<br/><br/>The Web offers the tools to reach out and be a world citizen/worker. I ran across my phone bill from a few years ago, I cut US $2k off by using Skype. Before I became a blogger for Skype, I wrote an article for the ScienceWriters network urging my colleagues to use VOIP to expand their network of sources and also clients. Everyday now, some 350,000 people sign up for Skype. Over 400 million already have. It's amazing--and I'm not just saying that because I am a blogger for Skype.<br/><br/>Seemingly silly social media, such as Twitter, are and will become increasingly important. Some people I run into seem to feel they already take up too much time doing e-mail. They reject doing Facebook and Twitter. I even talk to young people who feel that way. Some see it as an invasion of privacy. They feel teched out. Get over it. I say give it a try. Facebook and Twitter can be about more than what you had for breakfast.<br/><br/>LTB: As a freelancer, can you plan your work for longer time periods (I mean more than 3 to 6 months)?<br/><br/>HW: I do the best I can to structure my time so I can count on a revenue stream. So far, I can rely on Skype and Medill as a base. Everything else is gravy. It's hard to know what's coming. I am helping develop a new magazine now, but this won't last. I have a line on a potentially lucrative consulting job in the fall, but I can't count on it. Clients come and go.<br/><br/>LTB: What was the most important for you to have a successful transition from employee to freelancer?<br/><br/>HW: In the US, make sure you have health insurance. I had the advantage of working in the field for decades. It would be different if I were totally starting out. Networking is vital. Some writers I know are great networking to cover a beat, but may don't have a clue on networking in the writing business world. They're going to have to learn.<br/><br/>When I decided I was leaving my job, I wrote down every e-mail address I had and sent out notes announcing my departure. The networking helped. Contacts came up with leads that turned into jobs.<br/><br/>I had belong to the National Association of Science Writers for 25 years. Never went to a meeting. Until last year. I walked away from my first meeting with a lead on the teaching position at Medill. I have joined several other groups and have attended meetings. You never know when one thing leads to another.<br/><br/>Help others find work. They may return the favor.<br/><br/>Use LinkedIn, Facebook and Twitter.<br/><br/>LTB: With your experience now, at what stage of a career would you recommend younger people to start their preparation to become a freelancer?<br/><br/>HW: Maybe we'll all be freelancers in the future. The 27-year job like I held seems to be disappearing with the daily dead tree newspaper.<br/><br/>On my first job in 1970, we used manual typewriters. Soon, we switched to IBM electrics. We typed a code on the stories and old-fashioned pressmen--the guys in the paper hats--fed the stories into scanners. We became the first computer generation.<br/><br/>But I'll never forget one of the columnists, Gil, couldn't adjust to the electric typewriter. He became frustrated. He gave up. He was like the film stars of the silent era who couldn't make the transition to the talkies. Gil retired in his '50s.<br/><br/>Gil is an object lesson for us all. Unless you just want to go fishing full-time--maybe for good for you, but not me: Be open to change. Embrace new technology. Be excited about what you're doing. Keep learning.<br/><br/>LTB: But where is journalism going these days? I see that classical media is shaking and journals are changing habits. So journalism is changing too. But my specific question here is, how will scientific publishing change? We see the waves of Open Access Journals Movement (PLOS and the others) but these are still good classical peer reviewed journals. Do you see any novel type of media that could replace journals? What is your opinion about JOVE (Jounal of Visualized Experiments). Could this be a new type of publishing? Could someone build a scientific blog and stop counting the Impact Factors but start to count visits and links? Could this kind of change happen?<br/>Too many questions again.<br/>Maybe let's summarize it in one single question:<br/>Where is scientific journalism going?<br/><br/>HW: Good question. But I am no visionary on scientific publications, let alone lay publications.<br/><br/>Maybe 10 years ago, I saw a collision coming. I saw how all lay media were becoming one. I heard audio clips on the Wall Street Journal site, and saw text stories on TV websites and now video clips on print news sites.<br/><br/>I even have become a bit of a video reporter. I always try to do video interviews via webcam for my stories on the Skype blog. People have different ways of accessing/inputting information. The more choices you can give them, the better your odds of reaching them.<br/><br/>I also took a visual story telling class to learn what I could about doing video for the Web.<br/><br/>That said, I am not familiar with the video project you mentioned. I'll have to take a look.<br/><br/>All media seem to be going through a revolution, seeking new models and new ways to find and pay their way.<br/><br/>The students in my science/health writing class at Medill/Northwestern—the next generation of media— are required to do text stories. But also to do video and interactive graphics. They need to be flexible and masters or at least knowledgeable about all media.<br/><br/>I hope text--at least web-based articles, if not dead trees--will survive. Video story telling can be compelling. But I'd be skeptical that really complex stories can be told that way. Maybe I sound like a sentimental traditionalist, but I hope the word survives in print or on the web, for lay as well as scientific publications.<br/><br/>Thank you very much for your time and effort to share your ideas with us!<br/><br/>-----------------------<br/><br/>You can have a deeper insight in Howard's work on the following sites: <a title="Sweet Home Chicago" href="http://www.gapersblock.com/detour/so_long_suntimes/" target="_blank">here</a>, <a title="businessweek" href="http://www.businessweek.com/bwdaily/dnflash/content/mar2008/db20080314_121054.htm?campaign_id=rss_topStories" target="_blank">here</a>, <a title="Machu-Pichu" href="http://blog.orbitz.com/orbitz_blog/2008/08/machu-picchu-cu.html" target="_blank">here</a> and <a title="Skype" href="http://share.skype.com/sites/us/2009/03/post_9.html" target="_blank">here</a>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-44487699728031140592009-04-12T20:25:00.000+02:002010-07-24T16:55:08.837+02:00When I first heard about the Polymerase Chain Reaction my first association was with the <a href="http://en.wikipedia.org/wiki/Nuclear_chain_reaction">atomic bomb chain reaction</a>. You know probably from your studies: the labile Uranium if receives a neutron it transformed to a stable Uranium isotope while several new neutrons are released. If these newly release neutrons meet novel labile Uranium atoms the reaction is amplified, more and more neutrons will be released until the system if is lost from control explodes in the form of an atomic bomb. If the reaction is under control we can produce heat and through this electricity in an electric plant, if the critical mass of the labile isotope is ignited with a neutron beam, it will explode.<br/><br/>You can have two excellent representations of the chain reaction below:<br/><br/>The chain reaction<br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/ORqc1x3_Evg&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/ORqc1x3_Evg&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/FQGtpo2IUxA&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/FQGtpo2IUxA&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>But this is a blog about techniques in the molecular biology lab, so we will not deal with the fission chain reaction but we will see how a similar type of amplified reaction is produced with the DNA by specific enzymes in a reaction tube. The enzymes that can do a chain reaction are the <a href="http://en.wikipedia.org/wiki/Polymerase" target="_blank">Polymerases</a>.<br/><br/>What is the function of polymerases? We have them in each of our cells. They do the most basic reaction that keeps life going on from the start of the very first organism ever. They are duplicating in a semi-conservative way the DNA in order to allow the transmission of the genetic information during cell division.<br/><br/>You can have an animation about DNA replication below.<br/><br/>What is the Polymerase doing?<br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/teV62zrm2P0&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/teV62zrm2P0&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>The two DNA strands are connected by hydrogen bonds and code for the same information by the A:T and C:G base pairing. The two strands are anti-parallel if we look to the double strand from one direction one of the strands will be in 5'-3' direction and the other vice-versa in 3'-5' direction. As an important rule we have to know that in nature all polymerases are doing the DNA synthesis in the 5'-3' direction. The strand that can be replicated according to this rule is the leading strand. The other one is called lagging strand. The problem is that both strands have to replicated in by the same protein complex! But how can one single Replication complex produce the leading strand and the lagging strand in the same time ? We arrived to the problem of the the <a href="http://en.wikipedia.org/wiki/Okazaki_fragment" target="_blank">Okazaki fragments</a>.<br/><br/>In the animation below you can find a good representation about how a single Replication complex can do the synthesis of the two different strands.<br/><br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/4jtmOZaIvS0&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/4jtmOZaIvS0&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>In order to speak about PCR we have to go out to trip to check some Geysers.<br/><br/>So let us check first the big <a href="http://en.wikipedia.org/wiki/Steamboat_Geyser" target="_blank">Steamboat Geyser.<br/></a><br/><br/>Yellowstone Steamboat Geyser<br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/LSrQzv2oMw8&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/LSrQzv2oMw8&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>If we go closer to one of the hot springs we might see that the water is "living", there are some algae, micro-organisms in this water. Let's have a look:<br/><br/>The Yellowstone Hot Springs<br/><br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/oLAnm8kKz40&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/oLAnm8kKz40&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>These micro-organisms are living in really hot water. But if they are living, they should replicate, and if they replicate, they should have DNA polymerases!<br/><br/>These micro-organisms were isolated and one of them, called Thermo aquaticus (sometimes named <a href="http://en.wikipedia.org/wiki/Thermophilus_aquaticus" target="_blank">Thermophilus aquaticus</a>) became really famous. It has a polymerase that is used in vast majority of the in vitro DNA replication processes and in PCR.<br/><br/>I am sure you all have an idea already about PCR. We put all reagents needed for a DNA replication in a tube and reproduce the normal DNA replication process. So what do we need? We need a DNA template an oligonucleotide as a primer, the building blocks of the DNA (dATP, dTTP, dCTP, dGTP or in general dNTP -deoxi nucleotide tri phosphate), Mg and the Polymerase. If possible from Thermo aquaticus, which is called Taq.<br/><br/>There is one trick! This one trick was invented by <a href="http://en.wikipedia.org/wiki/Kary_Mullis" target="_blank">Kary Mullis</a> and he received Noble Prize for this single idea. The trick is, that we will not reproduce completely the natural reaction. We do not want to bother with leading strand and lagging strand and with all kind of Okazaki fragments and helicases and ligases.<br/><br/>The idea of Kary Mllis was that if you separate the double strand and design two oligos that will bind the two different strands but will look towards each other, than the product will be doubled. If you separate the strands by heating the solution to 95C you can repeat the reaction, and now you will have 4 copies. In the next run 8 copies and so on in each reaction you will have 2 on the power of the "cycle number" copies in a chain reaction fashion!!!<br/><br/>Let us have a really simple and good introduction to the whole procedure in the next two animations:<br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/QaWLJVGEFi8&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/QaWLJVGEFi8&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/-eOt_D4HtgQ&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/-eOt_D4HtgQ&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>Below you can find a more fancy animation of the same procedure:<br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/aGNYc8Etlc0&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/aGNYc8Etlc0&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>For this idea Mullis got the Noble Prize. His work changed completely the history of molecular biology. Let us check an interview with him about how he discovered PCR:<br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/xd4De47ldYs&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/xd4De47ldYs&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>What is the practical use of this whole method?<br/><br/>Amplifying DNA by PCR became one of the most widely used method in a molecular biology lab. You can use it for transferring DNA from one plasmid to a different one, to introduce mutations and even to measure the amount of a specific gene in a sample. By combining it with a Reverse Transcription reaction we can measure copy numbers of RNA molecules.<br/><br/>Below we can see an example of how it is used in criminal justice!<br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/fKTc1k2pK_k&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/fKTc1k2pK_k&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>In the next video you can see the workflow of DNA sequencing with a New Generation sequencing machine. What is remarkable here is that the designers of the instrument are skipping the cloning of the DNA fragments into plasmids and amplification of the plasmids by bacteria. They use micro reactors in the form of an emulsion PCR. One oligo is on a bead and the DNA binds the oligo. Each bead is fused with a small droplet that contains all the other reagents for the PCR. By this trick you will have a clonal amplification of the DNA fragments. One bead will contain on type of DNA and you skipped all bacterial work. The result is that you can sequence the whole human genome in a couple of weeks for less the 100k USD. Or you can sequence a bacteria in a day...<br/><br/>Emulsion PCR in the FLX sequencer workflow<br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/bFNjxKHP8Jc&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/bFNjxKHP8Jc&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object><br/><br/>At the end of this lesson, lets have some fun and see the celebration of the PCR!<br/><br/><object height="350" width="425"><br/> <param name="movie" value="http://www.youtube.com/v/x5yPkxCLads&hl=en&fs=1&rel=0"><br/> <param name="wmode" value="transparent"><br/> <embed src="http://www.youtube.com/v/x5yPkxCLads&hl=en&fs=1&rel=0;rel=0" type="application/x-shockwave-flash" wmode="transparent" height="350" width="425"><br/> </object>Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com0tag:blogger.com,1999:blog-6811136996857434306.post-25476604686125995152009-04-06T22:46:00.000+02:002010-07-24T16:55:06.862+02:00Restriction Enzyme ResourcesDear Colleagues,<br/><br/>Sometimes it is good to have links that cover a topic. This is why I have collected here a bunch of links that might be useful for you in your work.<br/><br/>You can have a good description of the methods used in restriction enzyme analysis here: <a href="http://www.methodbook.net/dna/restrdig.html" target="_blank">Methodbook.net</a><br/><br/>If you would like to use restriction enzymes for your work, you can find a list of links of the best known restriction enzyme providers below.<br/><br/><a href="http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/default.asp" target="_blank">New England Biolabs</a><br/><br/><a href="http://www.promega.com/guides/re_guide/">Promega</a><br/><br/><a href="https://www.roche-applied-science.com/benchmate/index.jsp" target="_blank">Roche Applied Science: Benchmate</a><br/><br/><a href="http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Restriction-Enzyme-Cloning.html" target="_blank">Invitrogen</a><br/><br/><a href="http://fermentas.com/catalog/re/index.html" target="_blank">Fermentas</a><br/><br/>If you want to start your work with these enzymes, please consult the protocol provided with the enzyme or check it at the website of the manufacturer.<br/><br/>Be sure you know what an <a href="http://en.wikipedia.org/wiki/Isoschizomer" target="_blank">isoschizomer</a> is, what <a href="http://en.wikipedia.org/wiki/Star_activity" target="_blank">star activity</a> is, or how you can make double digestion (details <a title="NEB" href="http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/double_digests.asp" target="_blank">here</a> and <a title="Fermentas" href="http://fermentas.com/doubledigest/index.html" target="_blank">here</a>).Balinthttp://www.blogger.com/profile/13879792030301762797noreply@blogger.com1