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

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<p><u>DNA  concentration assay</u></p>
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<p>pNP-Assay </p>
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<br>
<ol>
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<p style="text-align:left"><u>DNA  concentration assay</u></p>
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<br>
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<p style="text-align:left">pNP-Assay </p>
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<ol style="text-align:left">
 
   <li><u>Culture of DH5-alpha transformed with 808030 in pDG011</u></li>
 
   <li><u>Culture of DH5-alpha transformed with 808030 in pDG011</u></li>
 
   <li><u>Enzymatic essay (<strong>pNP assay</strong>) of the first transformation (of the  11/08/15): BBa_808030 and pDG011 plasmid in DH5</u><u>α</u></li>
 
   <li><u>Enzymatic essay (<strong>pNP assay</strong>) of the first transformation (of the  11/08/15): BBa_808030 and pDG011 plasmid in DH5</u><u>α</u></li>
 
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</ol>
<p>Gibson  Assembly : <br />
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 +
<br>
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<p style="text-align:left">Gibson  Assembly : <br />
 
   Enzymatic  digest of K936011, K936023 and K1392932 </p>
 
   Enzymatic  digest of K936011, K936023 and K1392932 </p>
<ol>
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<ol style="text-align:left">
 
   <li>Enzymatic digest using Pst I and Spe I </li>
 
   <li>Enzymatic digest using Pst I and Spe I </li>
 
   <li>Gel Migration of digested plasmids K936011, K936023,  K1392932. on a 0,8% Agarose Gel </li>
 
   <li>Gel Migration of digested plasmids K936011, K936023,  K1392932. on a 0,8% Agarose Gel </li>
 
   <li>Gel Purification </li>
 
   <li>Gel Purification </li>
 
</ol>
 
</ol>
<p><strong>Cluster 4A  Gibson assembly</strong><strong> </strong>:</p>
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<ol>
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<br>
 +
 
 +
<p style="text-align:left"><strong>Cluster 4A  Gibson assembly</strong><strong> </strong>:</p>
 +
<ol style="text-align:left">
 
   <li>Gibson Assembly </li>
 
   <li>Gibson Assembly </li>
 
   <li>PCR amplification of cluster 4A Assembly. </li>
 
   <li>PCR amplification of cluster 4A Assembly. </li>
 
   <li>Gel migration on 0,8% Agarose Gel. </li>
 
   <li>Gel migration on 0,8% Agarose Gel. </li>
 
</ol>
 
</ol>
<p>Gel Plan: </p>
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 +
<br>
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 +
<p style="text-align:left">Gel Plan: </p>
 
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Revision as of 21:28, 18 September 2015

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

    -

    Gel plan:


    Stain

    1

    2

    3

    4

    5

    6

    7

    8

    9

    10

    11

    Tube number

     

     

    A1

    A2

    A3

    A4

     

    B1

    B2

    B3

    B4

    Componants

    Ladder

     

     

     

     

     

     

     

     

     

     


     

    Concentration (ng/µL)

    OD(230nm)

    OD(260nm)

    OD(280nm)

    OD(260nm)/OD(280nm)

    OD(260nm)/OD(230nm)

    Cluster 4 start

    105

    0,3

    2,1

    1,1

    1,79

    2,40

    /936011

    55

    0

    1,1

    0,55

    1,99

    -

    936011/936023

    520

    4,325

    10,425

    5,825

    1,79

    4,38

    936023/

    47,5

    0

    0,925

    0,5

    1,89

    -

    Cluster 4 end

    130

    0,6

    2,625

    1,475

    1,79

    4,38


    DNA concentration assay


    pNP-Assay

    1. Culture of DH5-alpha transformed with 808030 in pDG011
    2. Enzymatic essay (pNP assay) of the first transformation (of the 11/08/15): BBa_808030 and pDG011 plasmid in DH5α

    Gibson Assembly :
    Enzymatic digest of K936011, K936023 and K1392932

    1. Enzymatic digest using Pst I and Spe I
    2. Gel Migration of digested plasmids K936011, K936023, K1392932. on a 0,8% Agarose Gel
    3. Gel Purification

    Cluster 4A Gibson assembly :

    1. Gibson Assembly
    2. PCR amplification of cluster 4A Assembly.
    3. Gel migration on 0,8% Agarose Gel.

    Gel Plan:


    Well Number

    1

    2

    3

    4

    5

    Contains

    DNA ladder

    No primers

    Primer for only

    Primer rev only

    Both primers



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