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Archive for the ‘transformation’ 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.

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