Team:UC Davis/protocols/transformation.html

From 2010.igem.org

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<li>Incubate at 37°C for 1 hour in a shaker. </li>
<li>Incubate at 37°C for 1 hour in a shaker. </li>
<li>Plate 200μL of each sample on <a href="https://2010.igem.org/Team:UC_Davis/protocols/luriabroth.html" class="help">nutrient agar plates.</a> (Be sure to choose the plates with the antibiotic that your E. Coli is resistant to.) </li>
<li>Plate 200μL of each sample on <a href="https://2010.igem.org/Team:UC_Davis/protocols/luriabroth.html" class="help">nutrient agar plates.</a> (Be sure to choose the plates with the antibiotic that your E. Coli is resistant to.) </li>
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<li>Spread the sample evenly on the plate by plating with sterilized glass beads. Empty the plate of glass beads afterwards.  </li>
+
<li>Spread the sample evenly by plating with sterilized glass beads. Empty the plate of glass beads afterwards.  </li>
<li>Incubate overnight at 37°C. </li>
<li>Incubate overnight at 37°C. </li>
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</ul>

Revision as of 00:17, 8 September 2010

E. Coli Transformation

Materials

You will need:

  • DH5α competent cells
  • polypropylene tubes
  • ice
  • ligation product
  • water bath
  • microtiter pipette & tips
  • luria broth
  • nutrient agar plates
  • Sterilized glass beads

Procedure

  • Thaw DH5α cells on ice for 10 minutes.
  • Transfer 50μL of DH5α cells to chill, sterile polypropylene tubes.
  • Transfer 1μL of ligation product into tubes.
  • Incubate on ice for 30 minutes.
  • Gently transfer tubes to 42°C water bath for 90s (for heat shock)
  • Transfer tubes back on ice for 2 minutes.
  • Add 800μL of luria broth into each tube.
  • Incubate at 37°C for 1 hour in a shaker.
  • Plate 200μL of each sample on nutrient agar plates. (Be sure to choose the plates with the antibiotic that your E. Coli is resistant to.)
  • Spread the sample evenly by plating with sterilized glass beads. Empty the plate of glass beads afterwards.
  • Incubate overnight at 37°C.

Purpose

To insert plasmids into E. Coli.

References
  • Notes by David Larsen

We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!

We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)

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