Difference between revisions of "Team:Pasteur Paris/Week 12"

Line 102: Line 102:
 
               <p></p><br><br><br>
 
               <p></p><br><br><br>
 
                
 
                
−
<p><em>Gibson  Assembly</em></p><br />
+
<p style="text-align:left"><em>Gibson  Assembly</em></p><br />
−
<ul>
+
<ul style="text-align:left>
 
<li>Gel  Migration of the PCR products (Cluster 2 and BBa_808014)</li>
 
<li>Gel  Migration of the PCR products (Cluster 2 and BBa_808014)</li>
 
<li>PCR  Amplification of intersequence 936011/936023 using Takara Ex Taq Polymerase.</li>
 
<li>PCR  Amplification of intersequence 936011/936023 using Takara Ex Taq Polymerase.</li>
Line 113: Line 113:
 
</ul>
 
</ul>
  
−
<p><em>NB  -Esterase assay </em></p>  
+
<p style="text-align:left><em>NB  -Esterase assay </em></p>  
  
−
<ul>
+
<ul style="text-align:left>
 
<li>Liquid culture of  the second transformation (of the 15/08/15): BBa_808030 and pDG011 plasmid in  DH5α </li>
 
<li>Liquid culture of  the second transformation (of the 15/08/15): BBa_808030 and pDG011 plasmid in  DH5α </li>
 
<li>MiniPrep of  BBa_808030 in the plasmid pDG011 in BAP 1</li>
 
<li>MiniPrep of  BBa_808030 in the plasmid pDG011 in BAP 1</li>
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</ul>
 
</ul>
  
−
<p><em>Gel Purification</em></p>
+
<p style="text-align:left><em>Gel Purification</em></p>
  
−
<p><u>Tubes components:</u></p>
+
<p style="text-align:left><u>Tubes components:</u></p>
 
<table border="1" cellspacing="0" cellpadding="0" width="480">
 
<table border="1" cellspacing="0" cellpadding="0" width="480">
 
   <tr>
 
   <tr>
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</table>
 
</table>
  
−
<ul>
+
<ul style="text-align:left>
 
   <li>Ligation of BBa_808030 and pDG011 </li>
 
   <li>Ligation of BBa_808030 and pDG011 </li>
 
   <li>Transformation in Chemically Competent DH5-⍺ cells </li>
 
   <li>Transformation in Chemically Competent DH5-⍺ cells </li>
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</ul>
 
</ul>
  
−
<p>&nbsp;</p>
+
 
−
<p>The bacteria did not grow  because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not  pSB1C3.<br />
+
<p style="text-align:left>The bacteria did not grow  because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not  pSB1C3.<br />
 
   - Bacterial culture  of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well. <br />
 
   - Bacterial culture  of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well. <br />
 
   - Midi-prep has been  done with the 50mL of BBa_J61047 culture. (dilution 1/1000) </p>
 
   - Midi-prep has been  done with the 50mL of BBa_J61047 culture. (dilution 1/1000) </p>
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   </tr>
 
   </tr>
 
</table>
 
</table>
−
<ul> <li>Preparation of YPD  specific media on specific media (1%  yeast extract, 2% Dextrose, 2% Peptone, 2% agar).</li>
+
<ul style="text-align:left> <li>Preparation of YPD  specific media on specific media (1%  yeast extract, 2% Dextrose, 2% Peptone, 2% agar).</li>
  
 
   <li>Gel Migration of BBa_J61047.</li>
 
   <li>Gel Migration of BBa_J61047.</li>
Line 188: Line 188:
 
   </ul>
 
   </ul>
 
    
 
    
−
   <ul>
+
   <ul style="text-align:left>
 
     <li>tube 1 «&nbsp;Miniprep XbaI/SpeI&nbsp;»&nbsp;:&nbsp;5uL 10X buffer cutsmart + 0,5uL  XbaI + 0,5uL SpeI + 38uL H2O +5uL ADN, digestion at 37°C during 1 hour.</li>
 
     <li>tube 1 «&nbsp;Miniprep XbaI/SpeI&nbsp;»&nbsp;:&nbsp;5uL 10X buffer cutsmart + 0,5uL  XbaI + 0,5uL SpeI + 38uL H2O +5uL ADN, digestion at 37°C during 1 hour.</li>
 
     <li>tube 2 «&nbsp;Midiprep XbaI/SpeI&nbsp;»&nbsp;: 5uL 10X buffer cut smart + 0,5uL XbaI +  0,5uL SpeI + 38uL H2O + 5uL ADN, digestion at 37°C during 1 hour</li>
 
     <li>tube 2 «&nbsp;Midiprep XbaI/SpeI&nbsp;»&nbsp;: 5uL 10X buffer cut smart + 0,5uL XbaI +  0,5uL SpeI + 38uL H2O + 5uL ADN, digestion at 37°C during 1 hour</li>
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</br>
 
</br>
  
−
<ul>
+
<ul style="text-align:left>
 
   <li>PCR Amplification of the  BBa_J61047 using Phusion Polymerase Master Mix. </li>
 
   <li>PCR Amplification of the  BBa_J61047 using Phusion Polymerase Master Mix. </li>
 
   <li>Gel Migration on a 0.8% agarose gel (See figure 01)<br />
 
   <li>Gel Migration on a 0.8% agarose gel (See figure 01)<br />
Line 208: Line 208:
 
</ul>
 
</ul>
  
−
<p><em>NB-Esterase Assay: </em></p>
+
<p style="text-align:left><em>NB-Esterase Assay: </em></p>
−
<ul>
+
<ul style="text-align:left>
 
<li>MiniPrep 808030 in the plasmid pDG011</li>
 
<li>MiniPrep 808030 in the plasmid pDG011</li>
 
<li>Gibson Assembly</li>
 
<li>Gibson Assembly</li>

Revision as of 21:06, 18 September 2015

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week 12

08/17 - 08/21




Gibson Assembly


    NB -Esterase assay

      Gel Purification


Tubes

Ladder

1st transformation

 

Components

 

GeneRuler 1kb DNA Ladder
(6x DNA loading dye)
3µL

Buffer
4µL
+
DNA
20µL
    The bacteria did not grow because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not pSB1C3.
    - Bacterial culture of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well.
    - Midi-prep has been done with the 50mL of BBa_J61047 culture. (dilution 1/1000)

     

    DNA Concentration (ng/µl)

    OD(260nm)

    OD(280nm)

    OD(260nm)/OD(280nm)

    BBa_J61047

    0,5

    0,011

    0,004

    2,57

    • tube 1 « Miniprep XbaI/SpeI » : 5uL 10X buffer cutsmart + 0,5uL XbaI + 0,5uL SpeI + 38uL H2O +5uL ADN, digestion at 37°C during 1 hour.
    • tube 2 « Midiprep XbaI/SpeI » : 5uL 10X buffer cut smart + 0,5uL XbaI + 0,5uL SpeI + 38uL H2O + 5uL ADN, digestion at 37°C during 1 hour
    • tube 3 « Miniprep NotI » : 5uL 10X buffer cutsmart + 0,5 NotI + 38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour
    • tube 4 « MiDiprep NotI » : 5uL 10X buffer cutsmart + 0,5 NotI + 38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour
    • tube 5 « Miniprep CTRL » as negative control : 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.
    • tube 6 « Midiprep CTRL » as negative control : 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.
    • MWT : « Quick-Load Purple 2-Log DNA Ladder (0.1-10.0 kb) NEB

      NB-Esterase Assay:

         

        Concentration (ng/µL)

        OD(230nm)

        OD(260nm)

        OD(280nm)

        OD(260nm)/OD(230nm)

        OD(260nm)/OD(280nm)

        Cluster_4_start

        30

        0,025

        0,575

        0,375

        1,50

        -

        /936011

        55

        0,4

        1,075

        0,575

        1,88

        2,65

        936011/936023

        7,5

        0,6

        0,125

        0,05

        2,41

        0,21

        936023/

        45

        0,575

        0,925

        0,625

        1,48

        1,61

        Cluster_4_end

        47,5

        0

        0,925

        0,575

        1,64

        -

^
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