Team:University of Ottawa/Transformation of Competent Ecoli

 

 color: white;} h1, h2, h3, h4, h5, h6 {color: white;}
 * 1) content {background: black;

  Untitled Document  

 Home  Welcome Announcements</a></li> </ul> The Team</a> <ul> Who We Are</a> <ul> Advisors</a></li> Undergrads</a></li> </ul> </li> What We've Done</a></li> Where We're From</a></li> Contact Us</a></li> </ul> </li> <li><a class="MenuBarItemSubmenu" href="http://2008.igem.org/Team:University_of_Ottawa/Project">The Project</a> <ul> <li><a href="http://2008.igem.org/Team:University_of_Ottawa/Project#Project_Overview">Overview</a></li> <li><a href="http://2008.igem.org/Team:University_of_Ottawa/Project#Pulsate_Gene_Expression">Expression</a></li> <li><a href="http://2008.igem.org/Team:University_of_Ottawa/Project#Cell-to-Cell_Communication">Communication</a></li> <li><a href="http://2008.igem.org/Team:University_of_Ottawa/Project#Oscillatory_Dynamics">Oscillation</a></li> <li><a href="http://2008.igem.org/Team:University_of_Ottawa/Project#Applications">Application</a></li> <li><a href="http://2008.igem.org/Team:University_of_Ottawa/Project#Journal_Club">Journal Club</a></li>

</ul> </li> <li><a href="http://2008.igem.org/Team:University_of_Ottawa/Parts">BioBricks</a></li> <li><a href="http://2008.igem.org/Team:University_of_Ottawa/Modeling">Modeling</a></li> <li><a class="MenuBarItemSubmenu" href="http://2008.igem.org/Team:University_of_Ottawa/Wet_Lab">Wet Lab</a> <ul> <li><a href="http://2008.igem.org/Team:University_of_Ottawa/Lab_Protocols">Lab Protocols</a></li> <li><a href="http://2008.igem.org/Team:University_of_Ottawa/WetWare">WetWare</a></li> </ul> <li><a href="http://2008.igem.org/Team:University_of_Ottawa/Notebook">Notebook</a></li> <li><a class="MenuBarItemSubmenu" href="http://2008.igem.org/Team:University_of_Ottawa/Sponsors">Sponsors</a> <ul> <li><a href="http://2008.igem.org/Team:University_of_Ottawa/Sponsors#Acedemic_Sponsors">Academic</a></li> <li><a href="http://2008.igem.org/Team:University_of_Ottawa/Sponsors#Research_Sponsors">Research</a></li> <li><a href="http://2008.igem.org/Team:University_of_Ottawa/Sponsors#Corporate_Sponsors">Corporate</a></li> </ul> </li>

</ul> <script type="text/javascript">

=Transformation of Competent Cells=

This protocol allows for fast and relatively efficient transformation of competent E. coli cells stored in TSS. All steps performed at the workbench should be accompanied by a flame for sterility. An alternate protocol that includes a heat shock treatment is also provided, but has not been shown to improve efficiency. <ol> <li> Thaw competent cells (DH5&alpha;, XL10, etc) stored at –80$deg;C on ice. <li> Set water bath or heat block to 42&deg;C, remove appropriate number of agar plates with correct antibiotic selection from the cold room to adjust to room temperature. If plate is wet, incubate at 37&deg;C incubator. <li> Add a maximum of 20 &mu;l of ligation mix (~50 ng of vector with insert) to a labeled tube containing the aliquoted competent cells (maintain cells on ice). An effort should be made to minimize the volume of DNA. <li> Add 100 &mu;L (lab grown) or 50 &mu;L (commercial) of competent cells to each tube. Mix by gently pipetting up and down with a wide bore pipet tip. <li> Set tubes on ice for 20 minutes. <li> Place tubes in the 42&deg;C water bath or heat block for 45 to 90s to allow entry of DNA by “heat shock”. <li> Set tubes back on ice for at least 5 min. <li> Add 900 &mu;l of LB broth to each tube of competent cells. <li> Incubate competent cells in shaker for 45 min at 37°C and 200 RPM. <li> Spin tubes for about 2 minute at 6,000 RPM (max setting) to pellet cells. <li> Discard all but 100μl of supernatant. <li> Resuspend cells in remaining supernatant by gently pipetting the mixture up and down, while trying not to create bubbles. <li> Pipette cell suspension onto LB agar plate with appropriate antibiotic selection conditions. Immerse spreader in 100% ethanol, pass it through flame and allow ethanol to burn off. Allow spreader to cool by making contact with the LB/agar away from the cell suspension with a back and forth movement for 1-2s. Spread cells evenly around with sterile spreader. <li> Allow cells to absorb into gel at room temperature on bench top for at least 30 min. <li> Incubate plates upside down at 37&deg;C overnight to prevent condensation from settling on the agar gel.