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Archive for March, 2011|Monthly archive page

Transformation of competent cells

In DNA, GFP, molecular biology, plasmid, Resources, Restriction Enzyme, transformation on March 16, 2011 at 4:53 pm

Scientific Background

Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells into which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation. It is important to note we have tested transformations of the distribution kit with this protocol. We have found that it is the best protocol. This protocol may be particularly useful if you are finding that your transformations are not working or yiedling few colonies.

In nature what happens is shown on the following two videos:

Overview

To see this in a nice lab demonstration tutorial about how transformation procedure is used, and why, watch this:

Materials

The demonstration of our iGEM protocol realized in summer 2009 is shown below:

Competent cells (we use DH5α)

DNA (this is a sample)

Ice

42°C water bath

37°C incubator

SOC (check for contamination!!)

Petri dishes with LB agar and appropriate antibiotic

Procedure

1. Start thawing the competent cells on crushed ice (we find this cells in the -70°C fridge)

2. Add 200μl competent cells and 2 or 5μl (50ng) DNA on ice

3. Incubate the cells for 30 minutes on ice

4. Heat shock at 42°C for 90 seconds water bath (not shaker)

5. Incubate for 5 minutes on ice

6. Add 200μl SOC broth (but sometimes not)

7. Shaker 2 hours at 37°C

8. (Sometimes centrifuge for 10 minutes at 10000 rpm and a few supernatant take int he dumb and suspendation the pellet)

9. Plate usually 60μl of the transformation or we make distribution 20μl and 200μl Petri dishes with agar and the appropriate antibiotic(s) with the part number, plasmid and antibiotic resistance

10. Incubate the plate at 37°C for 12-14 hours

Notes & troubleshooting

If you think another video demonstration would be needed, please go on to the next video:

More details about the procedure, with excellent links cand resource material can be found in the Molecular Biology Online Notebook.

References

1. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed.,1.25-1.28. Cold Spring Harbor Laboratory Press, Cold Spring harbor, NY, USA.

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The blog as a next generation book

In Blogging, Cool stuff on March 15, 2011 at 11:33 pm

Blogs are usually flowing, which makes them more like a diary or a newspaper. In this context, their information is difficult to structure. Tags, categories and search buttons can help but to have a really structured information on the blog is really difficult. On the other hand, blogs are extremly powerfull structures, that make possible the content management and combination of various types of information from text to videos. While wiki structure is well known to serve as a master structured content, it is difficult to work with beyond a basic structure.

Books, especially in science, are well structured texts. The author knows from beginning the sinopsis of the book, which migh slightly change during the writing itself, but more importantly, they are not written in the same order as they are placed in the book itself.

If we combine in a blog environment static pages and post pages, we can build a framework that allows us to make a new format of structured content, which is close to the classical book format but allows the combination of various media formats.

This format is A NEW BOOK TYPE that is more powerfull that any previous type of content.

This structure allows the writer to write sections in different time points but still to join them in one flow of information. This is a natural need of any writer who does not write simply a journal.

WordPress has a feature that allows the author to direct the reader to a specific page, e.g. the Contents while preserving the natural flow of individual posts. I suggest to use this combination for all blog authors who plan to build their blogs mainly on original content, and generate high quality resource centers in and around their blogs.

A very important feature of this blog- which is an experimental work- as you can see is that it is structured as a on-line book.  The Contents page servs not only to drive the attention of the reader, but also to FOCUS the author on the pages that are still missing. This platform is combining the flexibility of a blog and the riches given by web2.0 and multimedia tools, with the well know structured frame of a book. From this point of view, this stucture gives the best of both worlds. Information is well structured and rich, links and embeded videos are providing the reader context and real world simulation.

Enjoy!

Synthetic Biology to solve Global Health Challenges

In molecular biology on March 15, 2011 at 8:48 pm

The Bill & Melinda Gates Foundation is now accepting grant proposals for Round 7 of Grand Challenges Explorations, an initiative to encourage innovative and unconventional global health solutions. Applicants can be at any experience level; in any discipline; and from any organization, including colleges and universities, government laboratories, research institutions, non-profit organizations and for profit companies.

Grant proposals are being accepted online until May 19, 2011 on the following topics:

* Explore Nutrition for Healthy Growth of Infants and Children
* Apply Synthetic Biology to Global Health Challenges
* The Poliovirus Endgame: Create Innovative Ways to Accelerate, Sustain, and Monitor Eradication
* Create the Next Generation of Sanitation Technologies
* Design New Approaches to Cure HIV Infection
* Create Low-Cost Cell Phone-Based Solutions for Improved Uptake and Coverage of Childhood Vaccinations

Initial grants will be US $100,000 each, and projects showing promise will have the opportunity to receive additional funding of up to US $1 million.  Full descriptions of the new topics and application instructions are available at: http://www.grandchallenges.org/gce/

We are looking forward to receiving innovative ideas from around the world and from all disciplines. If you have a great idea, apply. If you know someone else who may have a great idea, please forward this message.

Thank you for your commitment to solving the world’s greatest health challenges.

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Bill & Melinda Gates Foundation

Guided by the belief that every life has equal value, the Bill & Melinda Gates Foundation works to help all people lead healthy, productive lives. In developing countries, it focuses on improving people’s health and giving them the chance to lift themselves out of hunger and extreme poverty. In the United States, it seeks to ensure that all people – especially those with the fewest resources – have access to the opportunities they need to succeed in school and life. Based in Seattle, the foundation is led by CEO Jeff Raikes and Co-chair William H. Gates Sr., under the direction of Bill and Melinda Gates and Warren Buffett.

Cell passaging video

In cell culture, iGEM, Lab equipment, molecular biology, Resources, sterile, video on March 9, 2011 at 9:48 pm

We think videos can help a lot for newcommers into the molecular biology laboratory.

One of the goals of our team (iGEM Debrecen 2010) was to generate high quality videos of basic lab protocols. All the protocolls can be viewed here but you can have an intro into Cell passaging righ now if you watch this video:

Why? This is the core question.

In Cool stuff, principles, Resources, video on March 9, 2011 at 4:40 pm

Why going in one particular direction if you can go in any direction? Why following someone, yes even in social media and by web2 tools? Only then comes the question “HOW” and at the very end “WHAT? Yes, the very last questions are WHAT TO DO. We have to know why we do something then how we should do that thing and at the end of course we need to know what exactly we shall do.

If you want to understand these principles, whatch the following video: