Team:Bordeaux/NotebookJune19

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(Difference between revisions)
(Created page with "{{:Team:Bordeaux/Tete}} <html> <h1>19<sup>th</sup> June </h1> <div class="type-exp"> Molecular biology</div> <br> <ul class="day-detail"> <li>PCR of our inserts 40 and 60 </li...")
 
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<ul class="day-detail">
<ul class="day-detail">
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<li>PCR of our inserts 40 and 60 </li>
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<li>PCR of our inserts 40 and 60 </li><br>
<li>Digestion of psB1C3 and our inserts 20-40-60 <BR><br>
<li>Digestion of psB1C3 and our inserts 20-40-60 <BR><br>
Concentration PCR 40 = 321,3ng/µl<BR>
Concentration PCR 40 = 321,3ng/µl<BR>
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  PCR 60 = 521,0ng/µl<br>
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<div style="margin-left:95px ;">   PCR 60 = 521,0ng/µl</div><br>
Concentration psB1C3 = 96,5ng/µl -> plate 4 kit 2014<br><br>
Concentration psB1C3 = 96,5ng/µl -> plate 4 kit 2014<br><br>
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digestion psB1C3 : </li>
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 +
Digestion psB1C3 : </li><br>
<table>
<table>
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   </tr>
   </tr>
</table>
</table>
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With vector = 5,18µL psB1C3 + 4,82µL H2O
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With vector = 5,18µL psB1C3 + 4,82µL H2O<br><br>
<li>Ligation</li>
<li>Ligation</li>
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So [plasmid] = 0,96ng/µl (via nanodrop)<br>
So [plasmid] = 0,96ng/µl (via nanodrop)<br>
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  <div style="margin-left:40px ;>  = 0,017 pmol/µl (via the website) </div><br>
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  <div style="margin-left:85px ;">  = 0,017 pmol/µl (via the website) <br>
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          <div style="margin-left:40px ;>  = 0,034 pmol/ 2µl </div><br>
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          = 0,034 pmol/ 2µl </div><br>
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So we need : 0,017*3=0,051pmol/µl  0,017 * 6 = 0,102pmol/ 2µl
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So we need : 0,017*3=0,051pmol/µl  0,017 * 6 = 0,102pmol/2µl
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<table>
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  <tr>
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    <th></th>
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    <th>20</th>
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    <th>40</th>
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    <th>60</th>
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  </tr>
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  <tr>
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    <th>Digestion</th>
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    <td>2µl</td>
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    <td>2µl</td>
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    <td>2µl</td>
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  </tr>
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  <tr>
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    <th>Insert</th>
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    <td>1,13µl</td>
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    <td>1,92µl</td>
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    <td>2,75µl</td>
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  </tr>
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  <tr>
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    <th>Buffer ligase 10X</th>
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    <td>1µl</td>
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    <td>1µl</td>
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    <td>1µl</td>
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  </tr>
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  <tr>
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    <th>T4 ligase</th>
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    <td>0,5µl</td>
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    <td>0,5µl</td>
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    <td>0,5µl</td>
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  </tr>
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  <tr>
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    <th>H2O qsp 10µl</th>
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    <td></td>
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    <td></td>
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    <td></td>
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  </tr>
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</table>
</ul>
</ul>
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<br><br>
<br><br>
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<div class="type-exp"> Purification</div>
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<div class="type-exp"> Design</div>
<br>
<br>

Latest revision as of 22:26, 17 October 2014

19th June

Molecular biology

  • PCR of our inserts 40 and 60

  • Digestion of psB1C3 and our inserts 20-40-60

    Concentration PCR 40 = 321,3ng/µl
    PCR 60 = 521,0ng/µl

    Concentration psB1C3 = 96,5ng/µl -> plate 4 kit 2014

    Digestion psB1C3 :

    • H2O (qsp 20µl) 7,5µl
    Buffer 2 2µl
    XbaI 0,25µl
    SpeI 0,25µl
    Vector 10µL
    With vector = 5,18µL psB1C3 + 4,82µL H2O

  • Ligation
    • We need to have a ratio [insert] = 3*[plasmid]
    We use the website promega
    • psB1C3 = 2270pb
    • VPGXG 20 = 436pb
    • VPGXG 40 = 736pb
    • VPGXG 60 = 1036pb


    So [plasmid] = 0,96ng/µl (via nanodrop)
    = 0,017 pmol/µl (via the website)
    = 0,034 pmol/ 2µl

    So we need : 0,017*3=0,051pmol/µl  0,017 * 6 = 0,102pmol/2µl
    20 40 60
    Digestion 2µl 2µl 2µl
    Insert 1,13µl 1,92µl 2,75µl
    Buffer ligase 10X 1µl 1µl 1µl
    T4 ligase 0,5µl 0,5µl 0,5µl
    H2O qsp 10µl


Purification

  • SDS-PAGE Tris-Glycine


Design

  • Design of oligo with DNA works (website)

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