IGEM:University of Debrecen:Transformation

 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.</o:p>

<p style="margin-top:4.8pt;margin-right:0in;margin-bottom:6.0pt;margin-left: 0in;line-height:18.0pt"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"> <span style="font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; color:black">Material</o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; line-height:18.0pt"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2">- <span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;mso-bidi-font-size: 11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family:&quot;Times New Roman&quot;; color:windowtext;text-decoration:none;text-underline:none">Resuspended DNA   <span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2">  <span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;">   <span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family: &quot;Times New Roman&quot;">(Resuspend well in 10ul dH20, pipette up and down several times, let sit for a few minutes</i>)</o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; line-height:18.0pt"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2">- <span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;mso-bidi-font-size: 11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family:&quot;Times New Roman&quot;; color:windowtext;text-decoration:none;text-underline:none">Competent cells   <span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2">  <span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">   <span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family: &quot;Times New Roman&quot;;color:black">(50ul per a transformation)</o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; line-height:18.0pt"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">-Ice</o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; line-height:18.0pt"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">-42º water bath</o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; line-height:18.0pt"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">-37º incubator</o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; line-height:18.0pt"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">-SOC (check for contamination!</i>)</o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; line-height:18.0pt"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">-Petri dishes with LB agar and appropriate antibiotic (two per a transformation)</o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; margin-left:.25in;line-height:18.0pt"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black"> </o:p>

<p class="MsoListParagraph" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; margin-left:0in;mso-add-space:auto;line-height:18.0pt"><span style="mso-bookmark: OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"> <span style="font-size:12.0pt; font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family:&quot;Times New Roman&quot;; color:black">Procedure</o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; margin-left:38.4pt;text-indent:-.25in;line-height:18.0pt;mso-list:l0 level1 lfo1; tab-stops:list .5in"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size: 10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family:Arial; color:black"> 1.<span style="font:7.0pt &quot;Times New Roman&quot;"> <span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">Start thawing the competent cells on crushed ice.</o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; margin-left:38.4pt;text-indent:-.25in;line-height:18.0pt;mso-list:l0 level1 lfo1; tab-stops:list .5in"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size: 10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family:Arial; color:black"> 2.<span style="font:7.0pt &quot;Times New Roman&quot;"> <span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">Add 50 µL of thawed competent cells and then 1 - 2 µL of the resuspended DNA to the labelled tubes. Make sure to keep the competent cells on ice.</o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; margin-left:38.4pt;text-indent:-.25in;line-height:18.0pt;mso-list:l0 level1 lfo1; tab-stops:list .5in"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size: 10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family:Arial; color:black"> 3.<span style="font:7.0pt &quot;Times New Roman&quot;"> <span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">Incubate the cells on ice for 30 minutes.</o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; margin-left:38.4pt;text-indent:-.25in;line-height:18.0pt;mso-list:l0 level1 lfo1; tab-stops:list .5in"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size: 10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family:Arial; color:black"> 4.<span style="font:7.0pt &quot;Times New Roman&quot;"> <span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells.<o:p></o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; margin-left:38.4pt;text-indent:-.25in;line-height:18.0pt;mso-list:l0 level1 lfo1; tab-stops:list .5in"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size: 10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family:Arial; color:black"> 5.<span style="font:7.0pt &quot;Times New Roman&quot;"> <span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">Incubate the cells on ice for 5 minutes.<o:p></o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; margin-left:38.4pt;text-indent:-.25in;line-height:18.0pt;mso-list:l0 level1 lfo1; tab-stops:list .5in"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size: 10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family:Arial; color:black"> 6.<span style="font:7.0pt &quot;Times New Roman&quot;"> <span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">Add 200 μl of SOC broth (make sure that the broth does not contain antibiotics and is not contaminated)<o:p></o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; margin-left:38.4pt;text-indent:-.25in;line-height:18.0pt;mso-list:l0 level1 lfo1; tab-stops:list .5in"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size: 10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family:Arial; color:black"> 7.<span style="font:7.0pt &quot;Times New Roman&quot;"> <span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. <span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">  <span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><b><span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family: &quot;Times New Roman&quot;;color:black">Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin. </b> <span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black"><o:p></o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; margin-left:38.4pt;text-indent:-.25in;line-height:18.0pt;mso-list:l0 level1 lfo1; tab-stops:list .5in"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size: 10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family:Arial; color:black"> 8.<span style="font:7.0pt &quot;Times New Roman&quot;"> <span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.<o:p></o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; margin-left:38.4pt;text-indent:-.25in;line-height:18.0pt;mso-list:l0 level1 lfo1; tab-stops:list .5in"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size: 10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;;mso-fareast-font-family:Arial; color:black"> 9.<span style="font:7.0pt &quot;Times New Roman&quot;"> <span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black">Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivate the antibiotic outside of the bacteria.<o:p></o:p>

<p class="MsoNormal" style="mso-margin-top-alt:auto;margin-bottom:1.2pt; line-height:18.0pt"><span style="mso-bookmark:OLE_LINK1"><span style="mso-bookmark:OLE_LINK2"><span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; mso-fareast-font-family:&quot;Times New Roman&quot;;color:black"><o:p> </o:p>

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<p style="margin-top:4.8pt;margin-right:0in;margin-bottom:6.0pt;margin-left: 0in;line-height:18.0pt"><span style="font-size:10.0pt;font-family:&quot;Arial&quot;,&quot;sans-serif&quot;; color:black"><o:p> </o:p>