http://www.openwetware.org/index.php?title=Transforming_chemically_competent_cells_protocol&feed=atom&action=history 2013-05-18T21:38:15Z Revision history for this page on the wiki MediaWiki 1.13.2 http://www.openwetware.org/index.php?title=Transforming_chemically_competent_cells_protocol&diff=368834&oldid=prev 2009-11-20T06:45:59Z

<p></p> <table style="background-color: white; color:black;"> <col class='diff-marker' /> <col class='diff-content' /> <col class='diff-marker' /> <col class='diff-content' /> <tr valign='top'> <td colspan='2' style="background-color: white; color:black;">←Older revision</td> <td colspan='2' style="background-color: white; color:black;">Revision as of 06:45, 20 November 2009</td> </tr> <tr><td colspan="2" class="diff-lineno">Line 1:</td> <td colspan="2" class="diff-lineno">Line 1:</td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>&lt;html&gt;</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>&lt;html&gt;</div></td></tr> <tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>&lt;h2&gt;Solutions/reagents:&lt;/h2&gt;&lt;ul type=&quot;circle&quot;&gt;&lt;li&gt;TSS cells&lt;/li&gt;&lt;li&gt;DNA&lt;/li&gt;&lt;li&gt;SOC stored at room temperature&lt;/li&gt;&lt;li&gt;plate made with appropriate antibiotic&lt;/li&gt;&lt;/ul&gt;&lt;h2&gt;Equipment:&lt;/h2&gt;&lt;ul type=&quot;circle&quot;&gt;&lt;li&gt;Incubator<del class="diffchange diffchange-inline">&lt;/li&gt;&lt;li&gt;Sterile 1.5-ml microcentrifuge tubes</del>&lt;/li&gt;&lt;/ul&gt;&lt;h2&gt;Steps:&lt;/h2&gt;&lt;ol&gt;&lt;p&gt;&lt;li<del class="diffchange diffchange-inline">&gt;Measure out TSS cells into sterile 1.5-ml microcentrifuge tube (1).&lt;br</del>&gt;Allow TSS cells to thaw on &lt;b&gt;&lt;font color=#357EC7&gt;ice&lt;/font&gt;&lt;/b&gt;.&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;<del class="diffchange diffchange-inline">Add &lt;font color=#357EC7&gt;</del>DNA<del class="diffchange diffchange-inline">&lt;/font&gt; to TSS cells</del>.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;(1µl of prepped plasmid is more than enough)&lt;/i&gt;&lt;/font&gt;&lt;br&gt;Mix solution by pipetting up and down several times.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Note: If you are adding small volumes (~1µl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Store the tube &lt;b&gt;&lt;font color=#357EC7&gt;on ice&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;30 mins&lt;/font&gt;&lt;/b&gt;.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Note: If you are in a rush, you can shorten this incubation time to 5-10 min.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Incubate at &lt;b&gt;&lt;font color=#357EC7&gt;42°C&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;30 secs&lt;/font&gt;&lt;/b&gt;.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Note: According to the original TSS paper and qualitative experience (JM), this step is completely optional and may actually reduce transformation efficiency.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Incubate on &lt;b&gt;&lt;font color=#357EC7&gt;&lt;b&gt;&lt;font color=#357EC7&gt;ice&lt;/font&gt;&lt;/b&gt;&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;2 mins&lt;/font&gt;&lt;/b&gt;.&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Add &lt;b&gt;&lt;font color=#357EC7&gt;1 ml&lt;/font&gt;&lt;/b&gt; of &lt;font color=#357EC7&gt;SOC&lt;/font&gt;.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;(2XYT and LB are also suitable, original paper suggests LB + 20mM glucose)&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Incubate at &lt;b&gt;&lt;font color=#357EC7&gt;37°C&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;1 hr&lt;/font&gt;&lt;/b&gt; with shaking at 200 rpm.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Note: Can also save some time here by reducing incubation to ~45 min.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Plate out &lt;b&gt;&lt;font color=#357EC7&gt;100 - 300 µl&lt;/font&gt;&lt;/b&gt; of SOC onto &lt;font color=#357EC7&gt;plate made with appropriate antibiotic&lt;/font&gt;.&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Incubate at &lt;b&gt;&lt;font color=#357EC7&gt;37°C&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;12 hrs&lt;/font&gt;&lt;/b&gt;(overnight).&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Save the rest of the transformants in liquid culture at 4°C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;/ol&gt;</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>&lt;h2&gt;Solutions/reagents:&lt;/h2&gt;&lt;ul type=&quot;circle&quot;&gt;&lt;li&gt;TSS cells&lt;/li&gt;&lt;li&gt;DNA&lt;/li&gt;&lt;li&gt;SOC stored at room temperature&lt;/li&gt;&lt;li&gt;plate made with appropriate antibiotic&lt;/li&gt;&lt;/ul&gt;&lt;h2&gt;Equipment:&lt;/h2&gt;&lt;ul type=&quot;circle&quot;&gt;&lt;li&gt;Incubator&lt;/li&gt;&lt;/ul&gt;&lt;h2&gt;Steps:&lt;/h2&gt;&lt;ol&gt;&lt;p&gt;&lt;li&gt;Allow TSS cells to thaw on &lt;b&gt;&lt;font color=#357EC7&gt;ice&lt;/font&gt;&lt;/b&gt;.&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;<ins class="diffchange diffchange-inline">Measure out </ins>DNA <ins class="diffchange diffchange-inline">into sterile 1.5-ml microcentrifuge tube (1)</ins>.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;(1µl of prepped plasmid is more than enough)&lt;/i&gt;&lt;/font&gt;&lt;br&gt;Mix solution by pipetting up and down several times.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Note: If you are adding small volumes (~1µl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Store the tube &lt;b&gt;&lt;font color=#357EC7&gt;on ice&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;30 mins&lt;/font&gt;&lt;/b&gt;.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Note: If you are in a rush, you can shorten this incubation time to 5-10 min.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Incubate at &lt;b&gt;&lt;font color=#357EC7&gt;42°C&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;30 secs&lt;/font&gt;&lt;/b&gt;.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Note: According to the original TSS paper and qualitative experience (JM), this step is completely optional and may actually reduce transformation efficiency.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Incubate on &lt;b&gt;&lt;font color=#357EC7&gt;&lt;b&gt;&lt;font color=#357EC7&gt;ice&lt;/font&gt;&lt;/b&gt;&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;2 mins&lt;/font&gt;&lt;/b&gt;.&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Add &lt;b&gt;&lt;font color=#357EC7&gt;1 ml&lt;/font&gt;&lt;/b&gt; of &lt;font color=#357EC7&gt;SOC&lt;/font&gt;.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;(2XYT and LB are also suitable, original paper suggests LB + 20mM glucose)&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Incubate at &lt;b&gt;&lt;font color=#357EC7&gt;37°C&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;1 hr&lt;/font&gt;&lt;/b&gt; with shaking at 200 rpm.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Note: Can also save some time here by reducing incubation to ~45 min.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Plate out &lt;b&gt;&lt;font color=#357EC7&gt;100 - 300 µl&lt;/font&gt;&lt;/b&gt; of SOC onto &lt;font color=#357EC7&gt;plate made with appropriate antibiotic&lt;/font&gt;.&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Incubate at &lt;b&gt;&lt;font color=#357EC7&gt;37°C&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;12 hrs&lt;/font&gt;&lt;/b&gt;(overnight).&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Save the rest of the transformants in liquid culture at 4°C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;/ol<ins class="diffchange diffchange-inline">&gt;&lt;p&gt;&lt;b&gt;TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :&lt;font color=#357EC7&gt;~ 13 hrs, 32 mins&lt;/font&gt;&lt;/b&gt;&lt;/p</ins>&gt;</div></td></tr> <tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>&lt;/html&gt;</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>&lt;/html&gt;</div></td></tr> <!-- diff generator: internal 2013-05-18 21:38:16 --> </table>

Vaishnavi Ananth http://www.openwetware.org/index.php?title=Transforming_chemically_competent_cells_protocol&diff=361511&oldid=prev 2009-10-23T06:59:18Z

<p>New page: &lt;html&gt; &lt;h2&gt;Solutions/reagents:&lt;/h2&gt;&lt;ul type=&quot;circle&quot;&gt;&lt;li&gt;TSS cells&lt;/li&gt;&lt;li&gt;DNA&lt;/li&gt;&lt;li&gt;SOC stored at room temperature&lt;/li&gt;&lt;li&gt;plate made with appropriate antibiotic&lt;/li&gt;&lt;/ul&gt;&lt;h2&gt;Equipment:...</p> <p><b>New page</b></p><div>&lt;html&gt;<br /> &lt;h2&gt;Solutions/reagents:&lt;/h2&gt;&lt;ul type=&quot;circle&quot;&gt;&lt;li&gt;TSS cells&lt;/li&gt;&lt;li&gt;DNA&lt;/li&gt;&lt;li&gt;SOC stored at room temperature&lt;/li&gt;&lt;li&gt;plate made with appropriate antibiotic&lt;/li&gt;&lt;/ul&gt;&lt;h2&gt;Equipment:&lt;/h2&gt;&lt;ul type=&quot;circle&quot;&gt;&lt;li&gt;Incubator&lt;/li&gt;&lt;li&gt;Sterile 1.5-ml microcentrifuge tubes&lt;/li&gt;&lt;/ul&gt;&lt;h2&gt;Steps:&lt;/h2&gt;&lt;ol&gt;&lt;p&gt;&lt;li&gt;Measure out TSS cells into sterile 1.5-ml microcentrifuge tube (1).&lt;br&gt;Allow TSS cells to thaw on &lt;b&gt;&lt;font color=#357EC7&gt;ice&lt;/font&gt;&lt;/b&gt;.&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Add &lt;font color=#357EC7&gt;DNA&lt;/font&gt; to TSS cells.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;(1µl of prepped plasmid is more than enough)&lt;/i&gt;&lt;/font&gt;&lt;br&gt;Mix solution by pipetting up and down several times.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Note: If you are adding small volumes (~1µl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Store the tube &lt;b&gt;&lt;font color=#357EC7&gt;on ice&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;30 mins&lt;/font&gt;&lt;/b&gt;.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Note: If you are in a rush, you can shorten this incubation time to 5-10 min.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Incubate at &lt;b&gt;&lt;font color=#357EC7&gt;42°C&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;30 secs&lt;/font&gt;&lt;/b&gt;.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Note: According to the original TSS paper and qualitative experience (JM), this step is completely optional and may actually reduce transformation efficiency.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Incubate on &lt;b&gt;&lt;font color=#357EC7&gt;&lt;b&gt;&lt;font color=#357EC7&gt;ice&lt;/font&gt;&lt;/b&gt;&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;2 mins&lt;/font&gt;&lt;/b&gt;.&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Add &lt;b&gt;&lt;font color=#357EC7&gt;1 ml&lt;/font&gt;&lt;/b&gt; of &lt;font color=#357EC7&gt;SOC&lt;/font&gt;.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;(2XYT and LB are also suitable, original paper suggests LB + 20mM glucose)&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Incubate at &lt;b&gt;&lt;font color=#357EC7&gt;37°C&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;1 hr&lt;/font&gt;&lt;/b&gt; with shaking at 200 rpm.&lt;br&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Note: Can also save some time here by reducing incubation to ~45 min.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Plate out &lt;b&gt;&lt;font color=#357EC7&gt;100 - 300 µl&lt;/font&gt;&lt;/b&gt; of SOC onto &lt;font color=#357EC7&gt;plate made with appropriate antibiotic&lt;/font&gt;.&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;Incubate at &lt;b&gt;&lt;font color=#357EC7&gt;37°C&lt;/font&gt;&lt;/b&gt; for &lt;b&gt;&lt;font color=#357EC7&gt;12 hrs&lt;/font&gt;&lt;/b&gt;(overnight).&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;p&gt;&lt;li&gt;&lt;font color = &quot;#800517&quot;&gt;&lt;i&gt;Save the rest of the transformants in liquid culture at 4°C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.&lt;/i&gt;&lt;/font&gt;&lt;br&gt;&lt;/li&gt;&lt;/p&gt;&lt;/ol&gt;<br /> &lt;/html&gt;</div>

Vaishnavi Ananth