Difference between revisions of "Template:BOKU-Vienna/GoldenGate iGEM Parts"

 
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<h2 id="golden-gate-cloning-protocol">Golden Gate Cloning Protocol</h2>
 
<h2 id="golden-gate-cloning-protocol">Golden Gate Cloning Protocol</h2>
 
<p>For the Golden Gate Cloning we use a concentration of 40 nM for the vector and 80 - 120 nM for the inserts. (Ratio 1:2 vector:insert or Ratio 1:3 vector:insert)
 
<p>For the Golden Gate Cloning we use a concentration of 40 nM for the vector and 80 - 120 nM for the inserts. (Ratio 1:2 vector:insert or Ratio 1:3 vector:insert)
−
The calculation was as following (With Example values, for interactive form click link below):</p>
+
The calculation was as following:</p>
−
<p><a href="https://2018.igem.org/Team:BOKU-Vienna/GoldenGate_Interactive">To interactive form</a></p>
+
 
<table class="table table-dark">
 
<table class="table table-dark">
 
   <thead>
 
   <thead>
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         <td> 68 </td>
 
         <td> 68 </td>
 
         <td> 132 </td>
 
         <td> 132 </td>
 +
    </tr>
 +
    <tr>
 +
        <td> GFP </td>
 +
        <td> 150 </td>
 +
        <td> 100 </td>
 +
        <td> 0.78374 </td>
 +
        <td> 39.187 </td>
 +
        <td> 2.5 </td>
 +
        <td> 80 </td>
 +
        <td> 120 </td>
 
     </tr>
 
     </tr>
 
   </tbody>
 
   </tbody>
 
</table>
 
</table>
 +
 +
<p></p><p>We used a standard assay for the assembly. From each diluted part we used 1 µL and mixed it with a Golden Gate MasterMix consists of BsaI or BbsI, T4 Ligase, CutSmart-Buffer, ATP and water.</p><p></p>
 +
<table class="table table-dark">
 +
  <thead>
 +
    <tr><th> Name </th><th> Volume [µL] </th><th> Additional </th></tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr><td> Backbone Plasmid </td><td> 1 </td><td></td></tr>
 +
    <tr><td> Insert 1 </td><td> 1 </td><td></td></tr>
 +
    <tr><td> - </td><td> - </td><td></td></tr>
 +
    <tr><td> CutSmart Buffer </td><td> 2 </td><td> Master Mix </td></tr>
 +
    <tr><td> ATP 10 mM    </td><td> 4 </td><td> Master Mix </td></tr>
 +
    <tr><td> BbsI </td><td> 2) </td><td> Master Mix </td></tr>
 +
    <tr><td> T4 Ligase 1:10 </td><td> 2.5 </td><td> Master Mix </td></tr>
 +
    <tr><td> dH2O </td><td> total volume of 20 µL </td><td> Master Mix </td></tr>
 +
  </tbody>
 +
</table>
 +
 +
<p></p><h3><a href="https://2018.igem.org/Team:BOKU-Vienna#golden-gate-thermocycler-protocol" id="golden-gate-thermocycler-protocol">Golden Gate Thermocycler Protocol</a></h3><p></p>
 +
<p></p><h4><a href="https://2018.igem.org/Team:BOKU-Vienna#protocol-1-4-hours" id="protocol-1-4-hours">Protocol 1 (4 hours)</a></h4><p></p>
 +
<table class="table-dark table">
 +
<tr>
 +
<th>Time [min]</th>
 +
<th>Temperatur</th>
 +
</tr>
 +
<tr>
 +
<td>1</td>
 +
<td>37 °C</td>
 +
</tr>
 +
<tr>
 +
<td>2.5</td>
 +
<td>16 °C </td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">45 repeats</td>
 +
</tr>
 +
<tr>
 +
<td>30</td>
 +
<td>37 °C </td>
 +
</tr>
 +
<tr>
 +
<td>5</td>
 +
<td>50 °C </td>
 +
</tr>
 +
<tr>
 +
<td>10</td>
 +
<td>80 °C </td>
 +
</tr>
 +
</table>
 +
 +
<p>After Golden Gate Assembly, the whole volume (20 uL) was transformed into competent E. coli cells.</p>

Latest revision as of 00:54, 18 October 2018

Golden Gate iGEM Parts

Send iGEM-Parts easyer and faster to the iGEM-Registry.

Because we worked during our work only with Golden Gate Systems, we had the problem how to send these parts to the iGEM - Registry. But we had an idea. What is if we combine Golden Gate Standard with the FC10 iGEM Standard?


The PCRs

First of all, each Part and also the Vector pSB1C3 has to tranform into GoldenGate Standards with BbsI cut sites.


As you can see in the picture 1 we designed the primer, for each parts in this way that these have the cutsites for the igemregistry and also for BbsI.

The Vector pSB1C3 got also the Cutsite for BbsI in this way, that the BbsI recognition site was removed at the assembly. 

       <a href="pSB1C3iGEM1.jpg" data-title="Primer Design for GoldenGate Igem Registry" data-toggle="lightbox">
           <img src="T--BOKU-Vienna--2018_pSB1C3iGEM1.jpg" alt="Primer Design for GoldenGate Igem Registry">
       </a>

       <a href="gToffGfpSequenceiGEM1.jpg" data-title="Primer Design for GoldenGate Igem Registry" data-toggle="lightbox">
           <img src="T--BOKU-Vienna--2018_gToffGfpSequenceiGEM1.jpg" alt="Primer Design for GoldenGate Igem Registry">
       </a>

And also for each part the primer was designed in this way, that they contain the cutsites for BbsI, EcoRI, NotI, XbaI, SpeI and PstI.


PCR Method

The Method for each PCR was a standard method for Q5 (from NEB)

<thead> 
 </thead>
 <tbody>

 </tbody>
Name Volume
Q5 High-Fidelity 2X Master Mix 25 µL
10 µM Forward Primer 2.5 µL
10 µM Reverse Primer 2.5 µL
Template DNA 2 µL
Nuclease free water 18 µL

Cleanup Protocol for PCR Product

For the cleaning of the PCR products we use a standard PCR/Gel Extraction kit. The Product was eluted in 50 µL Elution Buffer or Tris-HCl pH 8.

Golden Gate Cloning Protocol

For the Golden Gate Cloning we use a concentration of 40 nM for the vector and 80 - 120 nM for the inserts. (Ratio 1:2 vector:insert or Ratio 1:3 vector:insert) The calculation was as following:

<thead> </thead> <tbody> </tbody>
Plasmid-Name Nanodrop [µg/µL] Final Conc. [nM] Factor 1ng/ul -> 1nM Conc Stock [nM] Dilution DNA [µL] Tris-HCl [µL]
pSB1C3 150 40 0.78374 39.187 2.9 68 132
GFP 150 100 0.78374 39.187 2.5 80 120

We used a standard assay for the assembly. From each diluted part we used 1 µL and mixed it with a Golden Gate MasterMix consists of BsaI or BbsI, T4 Ligase, CutSmart-Buffer, ATP and water.

<thead> 
 </thead>
 <tbody>

 </tbody>
Name Volume [µL] Additional
Backbone Plasmid 1
Insert 1 1
- -
CutSmart Buffer 2 Master Mix
ATP 10 mM 4 Master Mix
BbsI 2) Master Mix
T4 Ligase 1:10 2.5 Master Mix
dH2O total volume of 20 µL Master Mix

<a href="https://2018.igem.org/Team:BOKU-Vienna#golden-gate-thermocycler-protocol" id="golden-gate-thermocycler-protocol">Golden Gate Thermocycler Protocol</a>

<a href="https://2018.igem.org/Team:BOKU-Vienna#protocol-1-4-hours" id="protocol-1-4-hours">Protocol 1 (4 hours)</a>

Time [min] Temperatur
1 37 °C
2.5 16 °C
45 repeats
30 37 °C
5 50 °C
10 80 °C

After Golden Gate Assembly, the whole volume (20 uL) was transformed into competent E. coli cells.