IGEM:University of Debrecen:Transformation

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<a name="OLE_LINK2"></a>Scientific background<o:p></o:p>

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.<o:p></o:p>

Material<o:p></o:p>

-Resuspended DNA (Resuspend well in 10ul dH20, pipette up and down several times, let sit for a few minutes)<o:p></o:p>

-Competent cells (50ul per a transformation)<o:p></o:p>

-Ice<o:p></o:p>

-42º water bath<o:p></o:p>

-37º incubator<o:p></o:p>

-SOC (check for contamination!)<o:p></o:p>

-Petri dishes with LB agar and appropriate antibiotic (two per a transformation)<o:p></o:p>

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Procedure<o:p></o:p>

1.    Start thawing the competent cells on crushed ice.<o:p></o:p>

2.    Add 50 µL of thawed competent cells and then 1 - 2 µL of the resuspended DNA to the labelled tubes. Make sure to keep the competent cells on ice.<o:p></o:p>

3.    Incubate the cells on ice for 30 minutes.<o:p></o:p>

4.    Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells.<o:p></o:p>

5.    Incubate the cells on ice for 5 minutes.<o:p></o:p>

6.    Add 200 μl of SOC broth (make sure that the broth does not contain antibiotics and is not contaminated)<o:p></o:p>

7.    Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.<o:p></o:p>

8.    Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.<o:p></o:p>

9.    Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivate the antibiotic outside of the bacteria.<o:p></o:p>

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