Difference between revisions of "Template:IONIS Paris/Notebook/11-09-15"

 
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<html>
 
<html>
 
<body>
 
<body>
−
<section id="cd-timeline" class="cd-container">
 
 
<div class="cd-timeline-block" id="11-09-15">
 
<div class="cd-timeline-block" id="11-09-15">
 
<div class="cd-timeline-content">
 
<div class="cd-timeline-content">
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</td>
 
</td>
 
<td>  
 
<td>  
−
1 µL
+
1 μL
 
</td>
 
</td>
 
<td>  
 
<td>  
−
1 µL
+
1 μL
 
</td>
 
</td>
 
<tr>
 
<tr>
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</td>
 
</td>
 
<td>  
 
<td>  
−
2 µL
+
2 μL
 
</td>
 
</td>
 
<td>  
 
<td>  
−
2 µL
+
2 μL
 
</td>
 
</td>
 
<tr>
 
<tr>
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</td>
 
</td>
 
<td>  
 
<td>  
−
15 µL
+
15 μL
 
</td>
 
</td>
 
<td>  
 
<td>  
−
15 µL
+
15 μL
 
</td>
 
</td>
 
</tr>
 
</tr>
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</td>
 
</td>
 
<td>  
 
<td>  
−
EcoRI 1 µL
+
EcoRI 1 μL
 
</td>
 
</td>
 
<td>  
 
<td>  
−
XbaI 1 µL
+
XbaI 1 μL
 
</td>
 
</td>
 
</tr>
 
</tr>
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</td>
 
</td>
 
<td>  
 
<td>  
−
SpeI 1 µL
+
SpeI 1 μL
 
</td>
 
</td>
 
<td>  
 
<td>  
−
PstI 1 µL
+
PstI 1 μL
 
</td>
 
</td>
 
</tr>
 
</tr>
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<i class="fa fa-angle-right"></i> 37°C, 1h <i class="fa fa-caret-right"></i> heat kill: 65°C, 20min<br>
 
<i class="fa fa-angle-right"></i> 37°C, 1h <i class="fa fa-caret-right"></i> heat kill: 65°C, 20min<br>
 
 
 +
</p>
 +
<div class="row">
 +
<div class="col-sm-6">
 +
<h5 style="color:rgba(255, 0, 0, 0.6);">Expected results</h5>
 +
<p align="center">
 +
<img src="https://static.igem.org/mediawiki/2015/f/f4/Expectedresultats-110915.png" alt="Expected results"  width=auto height="180">
 +
</p>
 +
</div>
 +
<div class="col-sm-6">
 +
<h5 style="color:rgba(255, 0, 0, 0.6);">Results </h5>
 +
<p align="center">
 +
<img src="https://static.igem.org/mediawiki/2015/2/29/Resultats-110915.png" alt="Results"  width=auto height="180">
 +
</p>
 +
</div> 
 +
</div>
 +
 +
 +
<center>
 +
<h4> <!-- Sous partie de l'experience -->
 +
Ligation
 +
</h4>
 +
</center>
 +
 +
 +
<table id="Mixligation">
 +
<caption valign="top" style="color:rgba(255, 0, 0, 0.6);font-weight: bold;"> Mix ligation preparation:</caption>
 +
<tr>
 +
<th>
 +
Tube
 +
</th>
 +
<th>
 +
pSB1C3-VVD YC
 +
</th>
 +
<th>
 +
pSB1C3-VVD YN
 +
</th>
 +
<th>
 +
pSB1C3
 +
</th>
 +
</tr>
 +
<tr>
 +
<td>
 +
<b>H<sub>2</sub>O MQ</b>
 +
</td>
 +
<td>
 +
2,2 μL
 +
</td>
 +
<td>
 +
3,4 μL
 +
</td>
 +
<td>
 +
1,8 μL
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<b>T4 ligase buffer</b>
 +
</td>
 +
<td>
 +
1 μL
 +
</td>
 +
<td>
 +
1 μL
 +
</td>
 +
<td>
 +
2 μL
 +
</td>
 +
</tr>
 +
 +
<tr>
 +
<td>
 +
<b>Insert</b>
 +
</td>
 +
<td>
 +
VVD YN, 4,1 µL
 +
</td>
 +
<td>
 +
VVD YC, 2,9 µL
 +
</td>
 +
<td>
 +
pDawn (28/07), 7,2 µL
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<b>Backbone</b>
 +
</td>
 +
<td>
 +
2,2 μL
 +
</td>
 +
<td>
 +
2,2 μL
 +
</td>
 +
<td>
 +
8 μL
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<b>T4 ligase</b>
 +
</td>
 +
<td>
 +
0,5 μL
 +
</td>
 +
<td>
 +
0,5 μL
 +
</td>
 +
<td>
 +
1 μL
 +
</td>
 +
</tr>
 +
</table>
 +
 +
<p>
 +
<i class="fa fa-angle-right"></i> 37°C, 30min <i class="fa fa-caret-right"></i> heat kill: 65°C, 20min<br>
 
 
−
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
 
 
</p>
 
</p>
 +
<p>
 +
<ul><li>
 +
Transformation of competent cells with pSB1C3-VVD YC + VVD YN and pSB1C3-VVD YN + VVD YC (1 µL or 4 µL have been used); 30°C all the weekend long
 +
</li></ul>
 +
 +
</p>
 +
 +
 +
 +
 +
 +
<center>
 +
<h4> <!-- Sous partie de l'experience -->
 +
Ligation pDawn Gblock
 +
</h4>
 +
</center>
 +
 +
 +
<table id="Mixligation">
 +
<caption valign="top" style="color:rgba(255, 0, 0, 0.6);font-weight: bold;"> Mix ligation preparation:</caption>
 +
<tr>
 +
<th>
 +
Tube
 +
</th>
 +
<th>
 +
pDawn I-II-III
 +
</th>
 +
<th>
 +
pDawn IV-V
 +
</th>
 +
</tr>
 +
 +
<tr>
 +
<td>
 +
<b>T4 ligase buffer</b>
 +
</td>
 +
<td>
 +
2 μL
 +
</td>
 +
<td>
 +
2,3 μL
 +
</td>
 +
</tr>
 +
 +
<tr>
 +
<td>
 +
<b>pDawn I</b>
 +
</td>
 +
<td>
 +
2,3 μL
 +
</td>
 +
<td>
 +
-
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<b>pDawn II</b>
 +
</td>
 +
<td>
 +
12 μL
 +
</td>
 +
<td>
 +
-
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<b>pDawn III</b>
 +
</td>
 +
<td>
 +
2,7 μL
 +
</td>
 +
<td>
 +
-
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<b>pDawn IV</b>
 +
</td>
 +
<td>
 +
-
 +
</td>
 +
<td>
 +
13,2 μL
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<b>pDawn V</b>
 +
</td>
 +
<td>
 +
-
 +
</td>
 +
<td>
 +
6,2 μL
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<b>T4 ligase</b>
 +
</td>
 +
<td>
 +
1 μL
 +
</td>
 +
<td>
 +
1 μL
 +
</td>
 +
</tr>
 +
</table>
 +
 +
<p>
 +
<i class="fa fa-angle-right"></i> Room temperature, 45min <i class="fa fa-caret-right"></i> heat kill: 65°C, 20min
 +
</p>
 
 
 +
<center>
 +
<h4>Purification of pSB1C3-pDawn</h4>
 +
</center>
 +
 +
 +
<blockquote>
 +
<i class="fa fa-angle-right"></i> 20 µL of ligation product + 80 µL of H<sub>2</sub>O MQ <br>
 +
<i class="fa fa-angle-right"></i> Add 1/10 (10 µL) of the volume of NaOAc 3M pH 5.2 <br>
 +
<i class="fa fa-angle-right"></i> Add 2,5 volumes (275 µL) of Ethanol 100% <br>
 +
<i class="fa fa-angle-right"></i> Mix and stored at -20°C for 1 hour at least <br>
 +
<i class="fa fa-angle-right"></i> Centrifuge 30 sec, 14000 rpm at 4°C <br>
 +
<i class="fa fa-angle-right"></i> Discard the supernatant <br>
 +
<i class="fa fa-angle-right"></i> Resuspend the pellet with 500 µL of ethanol 70%, invert up the tube several time <br>
 +
<i class="fa fa-angle-right"></i> Centrifuge 5 min, 14000 rpm at 4°C <br>
 +
<i class="fa fa-angle-right"></i> Discard the supernatant and let dry the pellet <br>
 +
<i class="fa fa-angle-right"></i> Resuspend the pellet with the right amount of water following the final concentration expected (20 µL here) <br>
 +
 +
</blockquote>
 +
 +
<center>
 +
<h4> <!-- Sous partie de l'experience -->
 +
Ligation pDawn Gblock
 +
</h4>
 +
</center>
 +
 +
 +
<table id="Mixligation">
 +
<caption valign="top" style="color:rgba(255, 0, 0, 0.6);font-weight: bold;"> Mix ligation preparation:</caption>
 +
<tr>
 +
<th>
 +
Tube
 +
</th>
 +
<th>
 +
pDawn I-II-III-IV-V
 +
</th>
 +
</tr>
 +
<tr>
 +
<td>
 +
<b>pDawn I-II-III</b>
 +
</td>
 +
<td>
 +
20 μL
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<b>pDawn IV-V</b>
 +
</td>
 +
<td>
 +
22,3 μL
 +
</td>
 +
</tr>
 +
 +
<tr>
 +
<td>
 +
<b>T4 ligase</b>
 +
</td>
 +
<td>
 +
1 μL
 +
</td>
 +
</tr>
 +
</table>
 +
 +
<p>
 +
<i class="fa fa-angle-right"></i> Room temperature, 45min <i class="fa fa-caret-right"></i> heat kill: 65°C, 20min
 +
</p>
 
 
 
 

Latest revision as of 10:26, 17 September 2015

Digestion

Mix digestion preparation:
Tube VVD YC VVD YN
H2O MQ 1 μL 1 μL
Buffer 2.1 2 μL 2 μL
DNA 15 μL 15 μL
Enzyme 1 EcoRI 1 μL XbaI 1 μL
Enzyme 2 SpeI 1 μL PstI 1 μL

37°C, 1h heat kill: 65°C, 20min

Expected results

Expected results

Results

Results

Ligation

Mix ligation preparation:
Tube pSB1C3-VVD YC pSB1C3-VVD YN pSB1C3
H2O MQ 2,2 μL 3,4 μL 1,8 μL
T4 ligase buffer 1 μL 1 μL 2 μL
Insert VVD YN, 4,1 µL VVD YC, 2,9 µL pDawn (28/07), 7,2 µL
Backbone 2,2 μL 2,2 μL 8 μL
T4 ligase 0,5 μL 0,5 μL 1 μL

37°C, 30min heat kill: 65°C, 20min

  • Transformation of competent cells with pSB1C3-VVD YC + VVD YN and pSB1C3-VVD YN + VVD YC (1 µL or 4 µL have been used); 30°C all the weekend long

Ligation pDawn Gblock

Mix ligation preparation:
Tube pDawn I-II-III pDawn IV-V
T4 ligase buffer 2 μL 2,3 μL
pDawn I 2,3 μL -
pDawn II 12 μL -
pDawn III 2,7 μL -
pDawn IV - 13,2 μL
pDawn V - 6,2 μL
T4 ligase 1 μL 1 μL

Room temperature, 45min heat kill: 65°C, 20min

Purification of pSB1C3-pDawn

20 µL of ligation product + 80 µL of H2O MQ
Add 1/10 (10 µL) of the volume of NaOAc 3M pH 5.2
Add 2,5 volumes (275 µL) of Ethanol 100%
Mix and stored at -20°C for 1 hour at least
Centrifuge 30 sec, 14000 rpm at 4°C
Discard the supernatant
Resuspend the pellet with 500 µL of ethanol 70%, invert up the tube several time
Centrifuge 5 min, 14000 rpm at 4°C
Discard the supernatant and let dry the pellet
Resuspend the pellet with the right amount of water following the final concentration expected (20 µL here)

Ligation pDawn Gblock

Mix ligation preparation:
Tube pDawn I-II-III-IV-V
pDawn I-II-III 20 μL
pDawn IV-V 22,3 μL
T4 ligase 1 μL

Room temperature, 45min heat kill: 65°C, 20min


11 Sept. 15