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

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<p style="text-align:left"><em>Gibson  Assembly</em></p><br />
 
<p style="text-align:left"><em>Gibson  Assembly</em></p><br />
−
<ul style="text-align:left>
+
<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>
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</ul>
 
</ul>
  
−
<p style="text-align:left><em>NB  -Esterase assay </em></p>  
+
<p style="text-align:left"><em>NB  -Esterase assay </em></p>  
  
−
<ul style="text-align:left>
+
<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 style="text-align:left><em>Gel Purification</em></p>
+
<p style="text-align:left"><em>Gel Purification</em></p>
  
−
<p style="text-align:left><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 style="text-align:left>
+
<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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−
<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 />
+
<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 style="text-align:left> <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>
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   </ul>
 
   </ul>
 
    
 
    
−
   <ul style="text-align:left>
+
   <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 style="text-align:left>
+
<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 style="text-align:left><em>NB-Esterase Assay: </em></p>
+
<p style="text-align:left"><em>NB-Esterase Assay: </em></p>
 
<ul style="text-align:left>
 
<ul style="text-align:left>
 
<li>MiniPrep 808030 in the plasmid pDG011</li>
 
<li>MiniPrep 808030 in the plasmid pDG011</li>

Revision as of 21:09, 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


  • Gel Migration of the PCR products (Cluster 2 and BBa_808014)
  • PCR Amplification of intersequence 936011/936023 using Takara Ex Taq Polymerase.
  • Gel Migration on 0,8% Agarose Gel
  • PCR Amplification of BBa_316003 using Takara Ex Taq Polymerase.
  • Gel Migration using 0,8% Agarose Gel
  • Spe I and Pst I Restriction Digest of the recieved plasmids containing BBa_K936011, BBa_K13932932, K936023.
  • Gel Purification of the digested plasmids K936011 and K1392932 using the QIAgen Gel extraction kit protocol.

NB -Esterase assay

  • Liquid culture of the second transformation (of the 15/08/15): BBa_808030 and pDG011 plasmid in DH5α
  • MiniPrep of BBa_808030 in the plasmid pDG011 in BAP 1
  • XbaI and PstI digestion of BBa_808030 and pDG011 (with the phosphatase step)

Gel Purification

Tubes components:


Tubes

Ladder

1st transformation

 

Components

 

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

Buffer
4µL
+
DNA
20µL
  • Ligation of BBa_808030 and pDG011
  • Transformation in Chemically Competent DH5-⍺ cells
  • Transformation in DH5-ɑ of the biobrick K808030 in pDG011
  • Transformation in DH5α cells of BBa_808030 insert and pDG011 vector
  • SpeI and Pst I Enzymatic Digest of BBa_K808030 with dephosphorylation.
  • Gel migration
  • Gel extraction of the PCR product of K808030 and pDG011.
  • Ligation of pDG011 and K808030.

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

  • Preparation of YPD specific media on specific media (1% yeast extract, 2% Dextrose, 2% Peptone, 2% agar).
  • Gel Migration of BBa_J61047.
  • NotI enzymatic digest of BBa_J61047 in pSB1A2 enzyme.
  • Gel Migration on a 0,8% Agarose Gel and the digestion hasn’t work.
  • XbaI and SpeI digestion of BBa_J61047.
  • 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

  • PCR Amplification of the BBa_J61047 using Phusion Polymerase Master Mix.
  • Gel Migration on a 0.8% agarose gel (See figure 01)
  • Culture of the plasmid pRS415 in SC_Leu at 30°C at 150rpm for about 72h.
  • PCR Amplification using the Phusion Polymerase of pRS415.

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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