In this self-guided slideshare presentation you will learn the basics of High Resolution Melt Analysis HRM, applications, important considerations, assay
Archive for 2011|Yearly archive page
Tons of Resources for High Resolution Melt Analysis
In molecular biology on August 28, 2011 at 10:33 amTransformation of competent cells
In DNA, GFP, molecular biology, plasmid, Resources, Restriction Enzyme, transformation on March 16, 2011 at 4:53 pmScientific 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.
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: 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 pmWe 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 pmWhy 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:
