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Archive for the ‘Resources’ Category

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.

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:

Melinda Gates about Polio and Coca-Cola

In Interview, principles, Resources, Uncategorized, video on January 24, 2011 at 7:29 am

Video project of the iGEM Debrecen Team

In Cool stuff, Resources, video on October 28, 2010 at 12:19 pm

Beside the whole project as beeing done, we completed a very nice video project.

See description here.

The protocolls can be found here.

The team wiki for the Debrecen iGEM team is here.

Good luck!

Designing a gene or “Gene design”

In animation, DNA, Gene design, iGEM, Polymerase Chain Reaction, Resources, Restriction Enzyme on June 8, 2010 at 9:19 pm

On this tutorial you can see an easy way to design oligos for gene synthesis, or in other words Gene design.

The description is based on the Instructional Videos from the iGEM website!

Gene design in less than 10 minutes!

Good luck!!!

Some usefull tools:

1. Gene design web page
2. Tools at DNA20
3. Tools at DNA Works
4. A Revese-Complement Tool
5. Gene designer a comprehensive tool to artificial gene design from DNA20 described here.

Restriction Enzyme Resources

In DNA, plasmid, Resources, Restriction Enzyme, Uncategorized on April 6, 2009 at 8:46 pm

Dear Colleagues,

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.

You can have a good description of the methods used in restriction enzyme analysis here: Methodbook.net

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.

New England Biolabs

Promega

Roche Applied Science: Benchmate

Invitrogen

Fermentas

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.

Be sure you know what an isoschizomer is, what star activity is, or how you can make double digestion (details here and here).