"
Team:Macquarie Australia/WetLab/Protocols/Heat
From 2014.igem.org
(Difference between revisions)
| Line 27: | Line 27: | ||
<div class="cont-out"> | <div class="cont-out"> | ||
<h3>Heat Shock</h3> | <h3>Heat Shock</h3> | ||
| - | < | + | <ul style="list-style-type: decimal;"> |
| + | <li>Obtain competent cells from -80<sup>o</sup>C.</li> | ||
| + | <li>Defrost gently on ice. 100µl is sufficient for 2 transformations.</li> | ||
| + | <li>Add 1-10µl of plasmid DNA/ ligation mix to each tube. Incubate on ice for 5 min.</li> | ||
| + | <li>Put the tubes in the 42<sup>o</sup>C water bath for 30 seconds, then back on ice for 2 min.</li> | ||
| + | <li>Add 200µl of SOC media to each tube, and incubate in the 37<sup>o</sup>C shaker for 10 min or up to 1h (10min for purified plasmid or 1h for ligation mix).</li> | ||
| + | <li>For each tube of cells, spread 50µl onto one LB plate with appropriate antibiotic, and 500µl onto a second plate, using aseptic technique. Place your plate upside-down in the 37<sup>o</sup>C incubator.</li> | ||
| + | </ul> | ||
| + | |||
</div> | </div> | ||
</section> | </section> | ||
</body> | </body> | ||
</html> | </html> | ||
Revision as of 05:01, 16 October 2014
Heat Shock
- Obtain competent cells from -80oC.
- Defrost gently on ice. 100µl is sufficient for 2 transformations.
- Add 1-10µl of plasmid DNA/ ligation mix to each tube. Incubate on ice for 5 min.
- Put the tubes in the 42oC water bath for 30 seconds, then back on ice for 2 min.
- Add 200µl of SOC media to each tube, and incubate in the 37oC shaker for 10 min or up to 1h (10min for purified plasmid or 1h for ligation mix).
- For each tube of cells, spread 50µl onto one LB plate with appropriate antibiotic, and 500µl onto a second plate, using aseptic technique. Place your plate upside-down in the 37oC incubator.
