Team:Freiburg/Notebook/lab crrna plasmid

From 2013.igem.org

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</p>
</p>
-
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/lab_effector"> Effector </a></p>
 
-
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/induction"> Induction </a> </p>
 
-
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/crrna"> Targeting </a></p>
 
-
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/method"> uniBAss </a></p>
 
-
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/modeling"> Modeling </a></p>
 
-
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/standardisation"> Standardisation </a></p>
 
-
 
-
<p class="second_order"> <a id="link" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_crrna_plasmid"> crRNA-Plasmid </a> </p>
 
-
<p class="second_order"> <a id="link" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_assembly_biobricks"> Assembly of Biobricks </a> </p>
 
 +
<p class="first_order"><a class="active" href="https://2013.igem.org/Team:Freiburg/Notebook/crrna"> Targeting </a></p>
 +
<p class="second_order_note"> <a id="link" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_multiple_targeting"> Multiple targeting </a> </p>
 +
<p class="second_order_note"> <a class="active" id="link" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_crrna_plasmid"> crRNA-Plasmid </a> </p>
 +
<p class="third_order"> <a href="#august"> August </a> </p>
 +
<p class="third_order"> <a href="#september"> September </a> </p>
 +
<p class="second_order_note"> <a id="link" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_gfp_reporter"> GFP-reporter-plasmid </a> </p>
 +
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/lab_effector"> Effector </a></p>
 +
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/induction"> Effector Control </a> </p>
 +
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/modeling"> Modeling </a></p>
 +
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/method"> uniBAss </a></p>
 +
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/standardisation"> Standardization </a></p>
 +
<p class="first_order"> <a id="link" href="https://2013.igem.org/Team:Freiburg/protocols"> Material and Methods </a> </p>
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-
<div id="main_contant" style="height:500px;">
+
<div id="main_contant_note">
<div id="h1">
<div id="h1">
-
crRNA-plasmid Notebook
+
crRNA-Plasmid Notebook
</div>
</div>
 +
<div id="tag">
 +
  <p id="h2"> Labbook RNA plasmid - pIG0021 </p>
 +
    <p> As a central part of our toolkit we need to clone a plasmid that contains a tracrRNA and a locus for inserting CRISPR RNAs into the PSB1C3 backbone. The general strategy is to amplify both loci from the px334a plasmid by Feng Zhang and combine them in the iGEM backbone.
 +
iGEM biobrick cutting sites will allow us to combine several targets with no big effort.
 +
    </p>
 +
</div>
 +
<div id="august">
 +
<p id="h2">
 +
August
 +
</p>
 +
</div>
 +
<div id="tag">
 +
  <h2> 28.08.13 </h2>
 +
    <h3> All primers arrived, PCRs for fragments. </h3>
 +
      <p> pU6:crRNA is amplified from px334a by PCR using the primers oIG0056 and the oU6:crRNA_fw primer. pH1:tracrRNA is amplified from pX334a by PCR suing the primers oH1:tracrRNA_fw and oH1:tracrRNA_rev. <br>
 +
  PCR was performed using the following master mix and reaction conditions.
 +
  In the same reaction the backbone of PSB1C3 was amplified using the primers oIG0037 and oIG0038.
 +
      </p>
 +
 +
</div>
 +
<div id="floatleft">
 +
  <table class="tabelle">
 +
  <tr>
 +
  <th> &micro;l </th>
 +
  <th> type </th>
 +
  </tr>
 +
  <tr>
 +
  <td> 10 </td>
 +
  <td> Q5-HF Reaction Buffer </td>
 +
  </tr>
 +
  <tr>
 +
  <td> 1 </td>
 +
  <td> Template </td>
 +
  </tr>
 +
  <tr>
 +
  <td> 1 </td>
 +
  <td> Primer1 </td>
 +
  </tr>
 +
  <tr>
 +
  <td> 1 </td>
 +
  <td> Primer2 </td>
 +
  </tr>
 +
  <tr>
 +
  <td> 4 </td>
 +
  <td> dNTPs </td>
 +
  </tr>
 +
  <tr>
 +
  <td> 1 </td>
 +
  <td> DMSO </td>
 +
  </tr>
 +
  <tr>
 +
  <td> 0.5 </td>
 +
  <td> Q5-HF Polymerase </td>
 +
  </tr>
 +
  <tr>
 +
  <td> Add to 50 </td>
 +
  <td> H<sub>2</sub>O </td>
 +
  </tr>
 +
  </table>
 +
  </div>
 +
  <div id="floatright">
 +
  <ul>
 +
<li> Annealing: 60&deg;C </li>
 +
<li> Elongation: 120 sec</li>
 +
<li> 24 cycles </li>
 +
  </ul>
 +
  </div>
 +
  <div id="tag">
 +
<p>Expected sizes are ~500bp for the RNA fragments and 2kb for the backbone. <br> Somebody must have mixed up TAE and ddH2o by preparating agarose. The gel was not really resolving the DNA. Nevertheless, the fragments contained the approximate size fragments. Bands were cut out and extracted using the Roche GelEx kit.
 +
<br> <br>  <b>Yields:</b> <br> pH1:tracrRNA: 2.2ng/ul <br> pU6:crRNA: 5.6 ng/ul <br> backbone: 22.1 ng/ul </p>
 +
</div>
 +
<br>
 +
 +
<div id="tag">
 +
<h3> Fusion PCR with all three fragments </h3>
 +
<p> As earlier approaches showed these fragments are problematic when using multi-fragment Gibson cloning or classical cloning respectively. Therefore the fragments were ligated by fusion PCR. </p>
 +
</div>
 +
<div id="floatleft">
 +
  <table class="tabelle">
 +
  <tr>
 +
  <th> &micro;l </th>
 +
  <th> type </th>
 +
  </tr>
 +
  <tr>
 +
  <td> 10 </td>
 +
  <td> Q5-HF Reaction Buffer </td>
 +
  </tr>
 +
  <tr>
 +
  <td> 3 </td>
 +
  <td> pH1:tracrRNA </td>
 +
  </tr>
 +
  <tr>
 +
  <td> 1 </td>
 +
  <td> PSB1C3 backbone </td>
 +
  </tr>
 +
  <tr>
 +
  <td> 3 </td>
 +
  <td> pU6:crRNA </td>
 +
  </tr>
 +
  <tr>
 +
  <td> 4 </td>
 +
  <td> dNTPs </td>
 +
  </tr>
 +
  <tr>
 +
  <td> 1 </td>
 +
  <td> DMSO </td>
 +
  </tr>
 +
  <tr>
 +
  <td> 0.5 </td>
 +
  <td> Q5-HF Polymerase </td>
 +
  </tr>
 +
  <tr>
 +
  <td> Add to 50 </td>
 +
  <td> H<sub>2</sub>O </td>
 +
  </tr>
 +
  </table>
 +
  </div>
 +
  <div id="floatright">
 +
<h4> first PCR </h4>
 +
  <ul>
 +
<li> Annealing: 60&deg;C </li>
 +
<li> Elongation: 180 sec</li>
 +
<li> 6 cycles </li>
 +
  </ul>
 +
  </div>
 +
    <div id="floatright">
 +
<h4> second PCR </h4>
 +
<p> 1ul of oH1:tracrRNA_fw and oIG0038 were added for complete amplification of linearised RNA plasmid. </p>
 +
  <ul>
 +
<li> Annealing: 60&deg;C </li>
 +
<li> Elongation: 180 sec</li>
 +
<li> 24 cycles </li>
 +
  </ul>
 +
  </div>
 +
<div id="tag">
 +
<p> This was done as a duplicate and put on a 1% agarose gel. The expected band runs at 3kb.</p>
 +
</div>
 +
<div>
 +
  <table class="gelpic">
 +
  <tr>
 +
<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/4/4e/1_crrna_plasmid_freiburg_13.jpg"> </td>
 +
  </tr>
 +
  <tr>
 +
<td> PCR aIG0021 </td>
 +
  </tr>
 +
</table>
 +
</div>
 +
<div id="tag">
 +
<p> Roth 1kb ladder was loaded. There are three bands visible, but only the most upper band fits the expected size. This band was cut out in both lanes and extracted by the Roche GelExtraction kit.
 +
<br> <b> Yields </b> <br> lane1: 9.6 ng/ul <br> lane2: 19.6 ng/ul <br> 
 +
</p>
 +
</div>
 +
 +
<div id="tag">
 +
<h3> Gibson assembly for pIG0021 </h3>
 +
<p> As the extracted linearised RNA plasmid contains complementary overhangs a 1-fragment Gibson was performed. To an aliquot of Gibson master mix 2.5 ul, respectively 5 ul of DNA was added and filled up with ddH2O to 5 ul. This mixture was incubated on 50° for 1 hour. Afterwards it was incubated for 3 minutes on ice and 3 minutes on RT.<br>
 +
5ul of this mixture was transformed into chemically competent <i> E.coli </i> and spread on chlorampheicol containing Agar plates. </p>
 +
</div>
 +
<br> <br>
 +
<div>
 +
<h2> 29.08.2013 </h2>
 +
<h4> Colony PCR </h4>
 +
<p> Clones were obtained. Colony PCR was performed to screen for potential positive clones. 15 clones were picked, transferred to Master Mix, transferred to Master Plate.
 +
</p>
 +
<div id="floatleft">
 +
  <table class="tabelle">
 +
  <tr>
 +
  <th> &micro;l </th>
 +
  <th> type </th>
 +
  </tr>
 +
  <tr>
 +
  <td> 2.5 </td>
 +
  <td>standard Taq buffer </td>
 +
  </tr>
 +
  <tr>
 +
  <td> 1 </td>
 +
  <td> oIG6017 </td>
 +
  </tr>
 +
  <tr>
 +
  <td> 1 </td>
 +
  <td> oIG6018</td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td> 2.5 </td>
 +
  <td> dNTPs </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td> 0.125 </td>
 +
  <td> Taq Polymerase </td>
 +
  </tr>
 +
  <tr>
 +
  <td> Add to 25 </td>
 +
  <td> H<sub>2</sub>O </td>
 +
  </tr>
 +
  </table>
 +
  </div>
 +
  <div id="floatright">
 +
  <ul>
 +
<li> Annealing: 60&deg;C </li>
 +
<li> Elongation: 80 sec</li>
 +
<li> 25 cycles </li>
 +
  </ul>
 +
  </div>
 +
<div>
 +
  <table class="gelpic">
 +
  <tr>
 +
<td> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/1/13/2_crrna_plasmid_freiburg_13.jpg"> </td>
 +
  </tr>
 +
  <tr>
 +
<td> cPCR pIG0021 </td>
 +
  </tr>
 +
</table>
 +
</div>
 +
<div id="tag">
 +
<p> Clones with bands at roughly 1.2 kb bands seem to be positive, but a little bit to high. Nevertheless, those clones were streak out on plates for mini prep.
 +
</p>
 +
</div>
 +
 +
<div>
 +
<h2> 30.08.2013 </h2>
 +
<p> Clones were prepped using the Promega mini prep kit. Test digest was performed using XbaI and Pst1-HF. Expected band sizes are 2kb and 0.9 kb. </p>
 +
</div>
 +
<div id="floatleft">
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> Substance </th>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Plasmid </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> CutSmart buffer</td>
 +
</tr>
 +
<tr>
 +
<td> 0,5 </td>
 +
<td> XbaI </td>
 +
</tr>
 +
<tr>
 +
<td> 0,5 </td>
 +
<td>  Pst1-HF </td>
 +
</tr>
 +
<tr>
 +
<td> 7 </td>
 +
<td> H2O </td>
 +
</tr>
 +
 +
</table>
 +
</div>
 +
<div id="floatright">
 +
<ul>
 +
<li> Incubation: 1.5 h at 37° C</li>
 +
</ul>
 +
</div>
 +
<h3> Results of test digest </h3>
 +
<div>
 +
<table class="image">
 +
<tr>
 +
<td> <img class="image" src="https://static.igem.org/mediawiki/2013/8/8a/3_crrna_plasmid_freiburg_13.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> 6 out of 8 colonies were positive. Clone 2 and 4 were sent for sequencing with oIG6017. </td>
 +
</tr>
 +
</table>
 +
</div>
 +
 +
 +
<div id="september">
 +
<p id="h2">
 +
September
 +
</p>
 +
</div>
 +
 +
<div>
 +
<h2> 04.09.2013 </h2>
 +
<p> after trouble with the sequencing company results are finally available. Clone 2 is sequenced as correct. RNA plasmid is correct as as planned. For further usage 2 re-transformations were performed to gain enough material for further experiments. </p>
 +
</div>
 +
 +
<div>
 +
<h2> 11.09.2013 </h2>
 +
<p> New plan: building multiple target RNA plasmids to avoid the additional cell stress, caused by multiple transfection with single target RNA plasmids. 1: VEGF target 4 + Bla target 2; 2: Bla target 3 + Bla target 4 </p>
 +
 +
<h3> Digest </h3>
 +
<div >
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> 2 </td>
 +
<td> VEGF target 2 plasmid / Bla target 3 plasmid </td>
 +
</tr>
 +
<tr>
 +
<td> 5 </td>
 +
<td> Cut Smart Buffer </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Spe </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Pst </td>
 +
</tr>
 +
<tr>
 +
<td> Add to 50&micro;l </td>
 +
<td> H<sub>2</sub>O </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div >
 +
<ul>
 +
<li> Temp.: 37&deg;C </li>
 +
<li> Incubation time: 2h </li>
 +
</ul>
 +
</div>
 +
 +
<div >
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> 2 </td>
 +
<td> Bla target 2 / Bla target 4 </td>
 +
</tr>
 +
<tr>
 +
<td> 5 </td>
 +
<td> Cut Smart Buffer </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Xba </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Pst </td>
 +
</tr>
 +
<tr>
 +
<td> Add to 50&micro;l </td>
 +
<td> H<sub>2</sub>O </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div >
 +
<ul>
 +
<li> Temp.: 37&deg;C </li>
 +
<li> Incubation time: 2h </li>
 +
</ul>
 +
</div>
 +
 +
<h3> Gelex </h3>
 +
<p>  Digest was load on gel; bands were cut out; Gelextraction </p>
 +
 +
<h3> Ligation </h3>
 +
<div >
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> 0,58 </td>
 +
<td> digested VEGF 2 target </td>
 +
</tr>
 +
<tr>
 +
<td> 16,67 </td>
 +
<td> digested Bla target 2 </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> T4 DNA Ligase </td>
 +
</tr>
 +
<tr>
 +
<td> 2 </td>
 +
<td> Buffer </td>
 +
</tr>
 +
<tr>
 +
<td> Add to 20&micro;l </td>
 +
<td> H<sub>2</sub>O </td>
 +
</tr>
 +
 +
</table>
 +
</div>
 +
<div>
 +
<ul>
 +
<li> Incubation for 15 minutes (RT) </li>
 +
</ul></div>
 +
 +
<div >
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> 1,15 </td>
 +
<td> digested Bla target 3 </td>
 +
</tr>
 +
<tr>
 +
<td> 12,82 </td>
 +
<td> digested Bla target 4 </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> T4 DNA Ligase </td>
 +
</tr>
 +
<tr>
 +
<td> 2 </td>
 +
<td> Buffer </td>
 +
</tr>
 +
<tr>
 +
<td> Add to 20&micro;l </td>
 +
<td> H<sub>2</sub>O </td>
 +
</tr>
 +
 +
</table>
 +
</div>
 +
<div>
 +
<ul>
 +
<li> Incubation for 15 minutes (RT) </li>
 +
</ul>
 +
</div>
 +
<p> → pIG4311, pIG4312 heat shock transfection in E. coli (according to protocol) <br>
 +
used: 5µl Ligationmix for 50µl TOP10 cells </p>
 +
 +
</div>
 +
<div>
 +
<h2> 13.09.2013 </h2>
 +
<p>Colony PCR</p>
 +
<div id="floatleft">
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> 2,5 </td>
 +
<td> Taq Buffer </td>
 +
</tr>
 +
<tr>
 +
<td>  </td>
 +
<td> Template </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Primer1 (oIG6017)</td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Primer2(oIG6018)</td>
 +
</tr>
 +
<tr>
 +
<td> 2.5 </td>
 +
<td> dNTPs </td>
 +
</tr>
 +
<tr>
 +
<td> 0.125 </td>
 +
<td> Taq Polymerase </td>
 +
</tr>
 +
<tr>
 +
<td> Add to 25 </td>
 +
<td> H<sub>2</sub>O </td>
 +
</tr>
 +
</table>
 +
</div>
 +
 +
<div><h3> Sequencing of positive Clones </h3></div>
 +
<p> Sequences positive </p> </div>
 +
 +
<div>
 +
<h2> 15.09.2013 </h2>
 +
<p> plan: multiple target RNA plasmid with VEGF target 4, Bla target 2, Bla target 3, Bla target 4 </p>
 +
<h3>Digest</h3>
 +
<div >
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> 2 </td>
 +
<td> pIG4311 </td>
 +
</tr>
 +
<tr>
 +
<td> 5 </td>
 +
<td> Cut Smart Buffer </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Spe </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Pst </td>
 +
</tr>
 +
<tr>
 +
<td> Add to 50&micro;l </td>
 +
<td> H<sub>2</sub>O </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div >
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> 2 </td>
 +
<td> pIG4312 </td>
 +
</tr>
 +
<tr>
 +
<td> 5 </td>
 +
<td> Cut Smart Buffer </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Xba </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Pst </td>
 +
</tr>
 +
<tr>
 +
<td> Add to 50&micro;l </td>
 +
<td> H<sub>2</sub>O </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<ul>
 +
<li> Incubation for 15 minutes (RT) </li>
 +
</ul>
 +
</div>
 +
<div >
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> 1,25 </td>
 +
<td> digested pIG4311 </td>
 +
</tr>
 +
<tr>
 +
<td> 3,63 </td>
 +
<td> digested pIG4312 </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> T4 DNA Ligase </td>
 +
</tr>
 +
<tr>
 +
<td> 2 </td>
 +
<td> Buffer </td>
 +
</tr>
 +
<tr>
 +
<td> Add to 20&micro;l </td>
 +
<td> H<sub>2</sub>O </td>
 +
</tr>
 +
 +
</table>
 +
</div>
 +
<div>
 +
<ul>
 +
<li> Incubation for 15 minutes (RT) </li>
 +
</ul>
 +
</div>
 +
<p> → pIG4313 heat shock transfection in E. coli (according to protocol) <br>
 +
used: 5µl Ligationmix for 50µl TOP10 cells </p>
 +
 +
</div>
 +
<div>
 +
<h2> 13.09.2013 </h2>
 +
<p>Colony PCR</p>
 +
<div id="floatleft">
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> 2,5 </td>
 +
<td> Taq Buffer </td>
 +
</tr>
 +
<tr>
 +
<td>  </td>
 +
<td> Template </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Primer1(oIG6017) </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Primer2(oIG6018) </td>
 +
</tr>
 +
<tr>
 +
<td> 2.5 </td>
 +
<td> dNTPs </td>
 +
</tr>
 +
<tr>
 +
<td> 0.125 </td>
 +
<td> Taq Polymerase </td>
 +
</tr>
 +
<tr>
 +
<td> Add to 25 </td>
 +
<td> H<sub>2</sub>O </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div><h3> Sequencing of positive Clones </h3></div>
 +
<p> Sequence positive </p> </div>
</div>
</div>
</body>
</body>
</html>
</html>

Latest revision as of 05:12, 28 October 2013


crRNA-Plasmid Notebook

Labbook RNA plasmid - pIG0021

As a central part of our toolkit we need to clone a plasmid that contains a tracrRNA and a locus for inserting CRISPR RNAs into the PSB1C3 backbone. The general strategy is to amplify both loci from the px334a plasmid by Feng Zhang and combine them in the iGEM backbone. iGEM biobrick cutting sites will allow us to combine several targets with no big effort.

August

28.08.13

All primers arrived, PCRs for fragments.

pU6:crRNA is amplified from px334a by PCR using the primers oIG0056 and the oU6:crRNA_fw primer. pH1:tracrRNA is amplified from pX334a by PCR suing the primers oH1:tracrRNA_fw and oH1:tracrRNA_rev.
PCR was performed using the following master mix and reaction conditions. In the same reaction the backbone of PSB1C3 was amplified using the primers oIG0037 and oIG0038.

µl type
10 Q5-HF Reaction Buffer
1 Template
1 Primer1
1 Primer2
4 dNTPs
1 DMSO
0.5 Q5-HF Polymerase
Add to 50 H2O
  • Annealing: 60°C
  • Elongation: 120 sec
  • 24 cycles

Expected sizes are ~500bp for the RNA fragments and 2kb for the backbone.
Somebody must have mixed up TAE and ddH2o by preparating agarose. The gel was not really resolving the DNA. Nevertheless, the fragments contained the approximate size fragments. Bands were cut out and extracted using the Roche GelEx kit.

Yields:
pH1:tracrRNA: 2.2ng/ul
pU6:crRNA: 5.6 ng/ul
backbone: 22.1 ng/ul


Fusion PCR with all three fragments

As earlier approaches showed these fragments are problematic when using multi-fragment Gibson cloning or classical cloning respectively. Therefore the fragments were ligated by fusion PCR.

µl type
10 Q5-HF Reaction Buffer
3 pH1:tracrRNA
1 PSB1C3 backbone
3 pU6:crRNA
4 dNTPs
1 DMSO
0.5 Q5-HF Polymerase
Add to 50 H2O

first PCR

  • Annealing: 60°C
  • Elongation: 180 sec
  • 6 cycles

second PCR

1ul of oH1:tracrRNA_fw and oIG0038 were added for complete amplification of linearised RNA plasmid.

  • Annealing: 60°C
  • Elongation: 180 sec
  • 24 cycles

This was done as a duplicate and put on a 1% agarose gel. The expected band runs at 3kb.

PCR aIG0021

Roth 1kb ladder was loaded. There are three bands visible, but only the most upper band fits the expected size. This band was cut out in both lanes and extracted by the Roche GelExtraction kit.
Yields
lane1: 9.6 ng/ul
lane2: 19.6 ng/ul

Gibson assembly for pIG0021

As the extracted linearised RNA plasmid contains complementary overhangs a 1-fragment Gibson was performed. To an aliquot of Gibson master mix 2.5 ul, respectively 5 ul of DNA was added and filled up with ddH2O to 5 ul. This mixture was incubated on 50° for 1 hour. Afterwards it was incubated for 3 minutes on ice and 3 minutes on RT.
5ul of this mixture was transformed into chemically competent E.coli and spread on chlorampheicol containing Agar plates.



29.08.2013

Colony PCR

Clones were obtained. Colony PCR was performed to screen for potential positive clones. 15 clones were picked, transferred to Master Mix, transferred to Master Plate.

µl type
2.5 standard Taq buffer
1 oIG6017
1 oIG6018
2.5 dNTPs
0.125 Taq Polymerase
Add to 25 H2O
  • Annealing: 60°C
  • Elongation: 80 sec
  • 25 cycles
cPCR pIG0021

Clones with bands at roughly 1.2 kb bands seem to be positive, but a little bit to high. Nevertheless, those clones were streak out on plates for mini prep.

30.08.2013

Clones were prepped using the Promega mini prep kit. Test digest was performed using XbaI and Pst1-HF. Expected band sizes are 2kb and 0.9 kb.

µl Substance
1 Plasmid
1 CutSmart buffer
0,5 XbaI
0,5 Pst1-HF
7 H2O
  • Incubation: 1.5 h at 37° C

Results of test digest

6 out of 8 colonies were positive. Clone 2 and 4 were sent for sequencing with oIG6017.

September

04.09.2013

after trouble with the sequencing company results are finally available. Clone 2 is sequenced as correct. RNA plasmid is correct as as planned. For further usage 2 re-transformations were performed to gain enough material for further experiments.

11.09.2013

New plan: building multiple target RNA plasmids to avoid the additional cell stress, caused by multiple transfection with single target RNA plasmids. 1: VEGF target 4 + Bla target 2; 2: Bla target 3 + Bla target 4

Digest

µl type
2 VEGF target 2 plasmid / Bla target 3 plasmid
5 Cut Smart Buffer
1 Spe
1 Pst
Add to 50µl H2O
  • Temp.: 37°C
  • Incubation time: 2h
µl type
2 Bla target 2 / Bla target 4
5 Cut Smart Buffer
1 Xba
1 Pst
Add to 50µl H2O
  • Temp.: 37°C
  • Incubation time: 2h

Gelex

Digest was load on gel; bands were cut out; Gelextraction

Ligation

µl type
0,58 digested VEGF 2 target
16,67 digested Bla target 2
1 T4 DNA Ligase
2 Buffer
Add to 20µl H2O
  • Incubation for 15 minutes (RT)
µl type
1,15 digested Bla target 3
12,82 digested Bla target 4
1 T4 DNA Ligase
2 Buffer
Add to 20µl H2O
  • Incubation for 15 minutes (RT)

→ pIG4311, pIG4312 heat shock transfection in E. coli (according to protocol)
used: 5µl Ligationmix for 50µl TOP10 cells

13.09.2013

Colony PCR

µl type
2,5 Taq Buffer
Template
1 Primer1 (oIG6017)
1 Primer2(oIG6018)
2.5 dNTPs
0.125 Taq Polymerase
Add to 25 H2O

Sequencing of positive Clones

Sequences positive

15.09.2013

plan: multiple target RNA plasmid with VEGF target 4, Bla target 2, Bla target 3, Bla target 4

Digest

µl type
2 pIG4311
5 Cut Smart Buffer
1 Spe
1 Pst
Add to 50µl H2O
µl type
2 pIG4312
5 Cut Smart Buffer
1 Xba
1 Pst
Add to 50µl H2O
  • Incubation for 15 minutes (RT)
µl type
1,25 digested pIG4311
3,63 digested pIG4312
1 T4 DNA Ligase
2 Buffer
Add to 20µl H2O
  • Incubation for 15 minutes (RT)

→ pIG4313 heat shock transfection in E. coli (according to protocol)
used: 5µl Ligationmix for 50µl TOP10 cells

13.09.2013

Colony PCR

µl type
2,5 Taq Buffer
Template
1 Primer1(oIG6017)
1 Primer2(oIG6018)
2.5 dNTPs
0.125 Taq Polymerase
Add to 25 H2O

Sequencing of positive Clones

Sequence positive

Retrieved from "http://2013.igem.org/Team:Freiburg/Notebook/lab_crrna_plasmid"