Team:Debrecen-Hungary/protocols/Transformation of competent cells

From 2010.igem.org

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(New page: ==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 call...)
 
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==Scientific Background==
==Scientific Background==
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1. Start thawing the competent cells on crushed ice (we find this cells in the -70°C fridge)
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1. Start thawing the competent cells on crushed ice  
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(we find this cells in the -70°C fridge)<Br>
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<div style="float: right;"> <html>
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<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/8fg_ldCMyW8" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/8fg_ldCMyW8" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object>
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</html></div><br>
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2. Add 200μl competent cells and 2 or 5μl (50ng) DNA on ice
 
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3. Incubate the cells for 30 minutes on ice
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2. Add 200μl competent cells and 2 or 5μl (50ng) DNA on ice<br><br><br>
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4. Heat shock at 42°C for 90 seconds water bath (not shaker)
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3. Incubate the cells for 30 minutes on ice<Br><br><br>
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5. Incubate for 5 minutes on ice
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4. Heat shock at 42°C for 90 seconds water bath (not shaker)<br><br><br>
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6. Add 200μl SOC broth (but sometimes not)
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5. Incubate for 5 minutes on ice<br>
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<div style="float: right;"> <html>
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<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/C_6LSQbNK3I" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/C_6LSQbNK3I" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object>
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</html></div><Br>
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7. Shaker 2 hours at 37°C
 
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8. (Sometimes centrifuge for 10 minutes at 10000 rpm and a few supernatant take int he dumb and suspendation the pellet)
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6. Add 200μl SOC broth (but sometimes not)<br><br><br>
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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
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7. Shaker 2 hours at 37°C<br><br><br>
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10. Incubate the plate at 37°C for 12-14 hours
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8. (Optional centrifuge for 10 minutes at 10000 rpm
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and remowe approx 85% of the supernatant.<br>
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Then sesuspend the pellet in the remaining broth.)<br><br><br>
 +
 
 +
9. Plate usually 60μl of the transformation
 +
or we make distribution 20μl and 200μl Petri dishes with agar
 +
<br>and the appropriate antibiotic(s) with the part number,
 +
plasmid and antibiotic resistance
 +
<div style="float: right;"> <html>
 +
<object style="height: 170px; width: 290px" ><param name="movie" value="http://www.youtube.com/v/s4suEpj26J4" ><param name="allowFullScreen" value="true" ><param name="allowScriptAccess" value="always" ><embed src="http://www.youtube.com/v/s4suEpj26J4" type="application/x-shockwave-flash" allowfullscreen="true" allowScriptAccess="always" width="290" height="170" ></object>
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</html></div><Br>
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10. Incubate the plate at 37°C for 12-14 hours<br><br><br><br><br><br><br>
==Notes & troubleshooting==
==Notes & troubleshooting==
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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.
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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==Other==
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==Links==
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[http://www.youtube.com/user/debrecenigem2010#p/u/7/8fg_ldCMyW8 Video I]
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[http://www.youtube.com/user/debrecenigem2010#p/u/6/C_6LSQbNK3I Video II]
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[http://www.youtube.com/user/debrecenigem2010#p/u/5/s4suEpj26J4 Video III]

Latest revision as of 16:16, 24 October 2010

Contents

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.

Overview

Materials

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. (Optional centrifuge for 10 minutes at 10000 rpm and remowe approx 85% of the supernatant.
Then sesuspend the pellet in the remaining broth.)


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

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.

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]

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