"
Team:Debrecen-Hungary/protocols/Transformation of competent cells
From 2010.igem.org
|
Scientific BackgroundTransformation 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. OverviewMaterialsCompetent 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
Procedure1. Start thawing the competent cells on crushed ice
(we find this cells in the -70°C fridge)
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
7. Shaker 2 hours at 37°C 8. (Optional centrifuge for 10 minutes at 10000 rpm
and remowe approx 85% of the supernatant. 9. Plate usually 60μl of the transformation
or we make distribution 20μl and 200μl Petri dishes with agar
10. Incubate the plate at 37°C for 12-14 hours Notes & troubleshootingReferences1. 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. Links[http://www.youtube.com/user/debrecenigem2010#p/u/7/8fg_ldCMyW8 Video I] [http://www.youtube.com/user/debrecenigem2010#p/u/6/C_6LSQbNK3I Video II] [http://www.youtube.com/user/debrecenigem2010#p/u/5/s4suEpj26J4 Video III] |
Team
