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

Line 121: Line 121:
 
       <td width="83" valign="top"><p align="center">OD(260nm)</p></td>
 
       <td width="83" valign="top"><p align="center">OD(260nm)</p></td>
 
       <td width="83" valign="top"><p align="center">OD(280nm)</p></td>
 
       <td width="83" valign="top"><p align="center">OD(280nm)</p></td>
       <td width="91" valign="top"><p align="center">OD(260nm)/OD(230nm)</p></td>
+
       <td width="91" valign="top"><p align="center">OD(260/230nm)</p></td>
       <td width="91" valign="top"><p align="center">OD(260nm)/OD(280nm)</p></td>
+
       <td width="91" valign="top"><p align="center">OD(260/280nm)</p></td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
Line 231: Line 231:
 
       <td width="92" valign="top"><p align="center">OD(260nm)</p></td>
 
       <td width="92" valign="top"><p align="center">OD(260nm)</p></td>
 
       <td width="92" valign="top"><p align="center">OD(280nm)</p></td>
 
       <td width="92" valign="top"><p align="center">OD(280nm)</p></td>
       <td width="92" valign="top"><p align="center">OD(260nm)/OD(280nm)</p></td>
+
       <td width="92" valign="top"><p align="center">OD(260/280nm)</p></td>
       <td width="92" valign="top"><p align="center">OD(260nm)/OD(230nm)</p></td>
+
       <td width="92" valign="top"><p align="center">OD(260/230nm)</p></td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
Line 312: Line 312:
 
     <tr>
 
     <tr>
 
       <td width="107" valign="top"><p>Contains</p></td>
 
       <td width="107" valign="top"><p>Contains</p></td>
       <td width="107" valign="top"><p>DNA   ladder</p></td>
+
       <td width="107" valign="top"><p>DNA ladder</p></td>
       <td width="107" valign="top"><p>No   primers</p></td>
+
       <td width="107" valign="top"><p>No primers</p></td>
       <td width="107" valign="top"><p>Primer   for only</p></td>
+
       <td width="107" valign="top"><p>Primer for only</p></td>
       <td width="107" valign="top"><p>Primer   rev only</p></td>
+
       <td width="107" valign="top"><p>Primer rev only</p></td>
       <td width="107" valign="top"><p>Both   primers</p></td>
+
       <td width="107" valign="top"><p>Both primers</p></td>
 
     </tr>
 
     </tr>
 
   </table>
 
   </table>
Line 332: Line 332:
 
   <table border="1" cellspacing="0" cellpadding="0" width="605">
 
   <table border="1" cellspacing="0" cellpadding="0" width="605">
 
     <tr>
 
     <tr>
       <td width="64" valign="top"><p>1/25   dilution</p></td>
+
       <td width="64" valign="top"><p>1/25 dilution</p></td>
       <td width="77" valign="top"><p>DNA   concentration (ng/µL)</p></td>
+
       <td width="77" valign="top"><p>DNA concentration (ng/µL)</p></td>
 
       <td width="77" valign="top"><p>OD(230nm)</p></td>
 
       <td width="77" valign="top"><p>OD(230nm)</p></td>
 
       <td width="77" valign="top"><p>OD(260nm)</p></td>
 
       <td width="77" valign="top"><p>OD(260nm)</p></td>
 
       <td width="77" valign="top"><p>OD(280nm)</p></td>
 
       <td width="77" valign="top"><p>OD(280nm)</p></td>
 
       <td width="77" valign="top"><p>OD(340nm)</p></td>
 
       <td width="77" valign="top"><p>OD(340nm)</p></td>
       <td width="77" valign="top"><p>OD(260nm)/OD(280nm)</p></td>
+
       <td width="77" valign="top"><p>OD(260/280nm)</p></td>
       <td width="77" valign="top"><p>OD(260nm)/OD(230nm)</p></td>
+
       <td width="77" valign="top"><p>OD(260/230nm)</p></td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
Line 421: Line 421:
 
   <tr>
 
   <tr>
 
     <td width="80" valign="top"><p>Contains</p></td>
 
     <td width="80" valign="top"><p>Contains</p></td>
     <td width="80" valign="top"><p>DNA   ladder</p></td>
+
     <td width="80" valign="top"><p>DNA ladder</p></td>
     <td width="80" valign="top"><p>Non   digested Plasmid</p></td>
+
     <td width="80" valign="top"><p>Non digested Plasmid</p></td>
     <td width="80" valign="top"><p>Digested   plasmid</p></td>
+
     <td width="80" valign="top"><p>Digested plasmid</p></td>
     <td width="80" valign="top"><p>No   primers</p></td>
+
     <td width="80" valign="top"><p>No primers</p></td>
     <td width="80" valign="top"><p>Primer   for only</p></td>
+
     <td width="80" valign="top"><p>Primer for only</p></td>
     <td width="80" valign="top"><p>Primer   rev only</p></td>
+
     <td width="80" valign="top"><p>Primer rev only</p></td>
     <td width="80" valign="top"><p>Both   primers</p></td>
+
     <td width="80" valign="top"><p>Both primers</p></td>
 
   </tr>
 
   </tr>
 
</table>
 
</table>
Line 438: Line 438:
 
   <tr>
 
   <tr>
 
     <td width="133" valign="top"><p>&nbsp;</p></td>
 
     <td width="133" valign="top"><p>&nbsp;</p></td>
     <td width="77" valign="top"><p>No   primers</p></td>
+
     <td width="77" valign="top"><p>No primers</p></td>
     <td width="71" valign="top"><p>Primer   for</p></td>
+
     <td width="71" valign="top"><p>Primer for</p></td>
     <td width="73" valign="top"><p>Primer   rev</p></td>
+
     <td width="73" valign="top"><p>Primer rev</p></td>
 
     <td width="45" valign="top"><p>&nbsp;</p></td>
 
     <td width="45" valign="top"><p>&nbsp;</p></td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td width="133" valign="top"><p>DNAse,   RNAse free water</p></td>
+
     <td width="133" valign="top"><p>DNAse, RNAse free water</p></td>
 
     <td width="77" valign="top"><p align="right">41,75</p></td>
 
     <td width="77" valign="top"><p align="right">41,75</p></td>
 
     <td width="71" valign="top"><p align="right">40,75</p></td>
 
     <td width="71" valign="top"><p align="right">40,75</p></td>
Line 451: Line 451:
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td width="133" valign="top"><p>10X   Ex Taq Buffer</p></td>
+
     <td width="133" valign="top"><p>10X Ex Taq Buffer</p></td>
 
     <td width="77" valign="top"><p align="right">5</p></td>
 
     <td width="77" valign="top"><p align="right">5</p></td>
 
     <td width="71" valign="top"><p align="right">5</p></td>
 
     <td width="71" valign="top"><p align="right">5</p></td>
Line 486: Line 486:
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td width="133" valign="top"><p>Ex   Taq Polymerase</p></td>
+
     <td width="133" valign="top"><p>Ex Taq Polymerase</p></td>
 
     <td width="77" valign="top"><p align="right">0,25</p></td>
 
     <td width="77" valign="top"><p align="right">0,25</p></td>
 
     <td width="71" valign="top"><p align="right">0,25</p></td>
 
     <td width="71" valign="top"><p align="right">0,25</p></td>
Line 543: Line 543:
 
   <tr>
 
   <tr>
 
     <td width="107" valign="top"><p>&nbsp;</p></td>
 
     <td width="107" valign="top"><p>&nbsp;</p></td>
     <td width="107" valign="top"><p>DNA   ladder</p></td>
+
     <td width="107" valign="top"><p>DNA ladder</p></td>
     <td width="107" valign="top"><p>No   primers</p></td>
+
     <td width="107" valign="top"><p>No primers</p></td>
     <td width="107" valign="top"><p>Primer   For</p></td>
+
     <td width="107" valign="top"><p>Primer For</p></td>
     <td width="107" valign="top"><p>Primer   Rev</p></td>
+
     <td width="107" valign="top"><p>Primer Rev</p></td>
 
     <td width="107" valign="top"><p>Extract</p></td>
 
     <td width="107" valign="top"><p>Extract</p></td>
 
   </tr>
 
   </tr>
Line 565: Line 565:
 
   <tr>
 
   <tr>
 
     <td width="133" valign="top"><p>&nbsp;</p></td>
 
     <td width="133" valign="top"><p>&nbsp;</p></td>
     <td width="77" valign="top"><p>No   primers</p></td>
+
     <td width="77" valign="top"><p>No primers</p></td>
     <td width="71" valign="top"><p>Primer   for</p></td>
+
     <td width="71" valign="top"><p>Primer for</p></td>
     <td width="73" valign="top"><p>Primer   rev</p></td>
+
     <td width="73" valign="top"><p>Primer rev</p></td>
 
     <td width="45" valign="top"><p>&nbsp;</p></td>
 
     <td width="45" valign="top"><p>&nbsp;</p></td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td width="133" valign="top"><p>DNAse,   RNAse free water</p></td>
+
     <td width="133" valign="top"><p>DNAse, RNAse free water</p></td>
 
     <td width="77" valign="top"><p align="right">41,75</p></td>
 
     <td width="77" valign="top"><p align="right">41,75</p></td>
 
     <td width="71" valign="top"><p align="right">40,75</p></td>
 
     <td width="71" valign="top"><p align="right">40,75</p></td>
Line 578: Line 578:
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td width="133" valign="top"><p>10X   Ex Taq Buffer</p></td>
+
     <td width="133" valign="top"><p>10X Ex Taq Buffer</p></td>
 
     <td width="77" valign="top"><p align="right">5</p></td>
 
     <td width="77" valign="top"><p align="right">5</p></td>
 
     <td width="71" valign="top"><p align="right">5</p></td>
 
     <td width="71" valign="top"><p align="right">5</p></td>
Line 606: Line 606:
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td width="133" valign="top"><p>DNA  Template</p></td>
+
     <td width="133" valign="top"><p>DNA Template</p></td>
 
     <td width="77" valign="top"><p align="right">1</p></td>
 
     <td width="77" valign="top"><p align="right">1</p></td>
 
     <td width="71" valign="top"><p align="right">1</p></td>
 
     <td width="71" valign="top"><p align="right">1</p></td>
Line 613: Line 613:
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
     <td width="133" valign="top"><p>Ex   Taq Polymerase</p></td>
+
     <td width="133" valign="top"><p>Ex Taq Polymerase</p></td>
 
     <td width="77" valign="top"><p align="right">0,25</p></td>
 
     <td width="77" valign="top"><p align="right">0,25</p></td>
 
     <td width="71" valign="top"><p align="right">0,25</p></td>
 
     <td width="71" valign="top"><p align="right">0,25</p></td>
Line 648: Line 648:
 
     <td width="121" valign="top"><p>OD(260nm)</p></td>
 
     <td width="121" valign="top"><p>OD(260nm)</p></td>
 
     <td width="121" valign="top"><p>OD(280nm)</p></td>
 
     <td width="121" valign="top"><p>OD(280nm)</p></td>
     <td width="121" valign="top"><p>OD(260nm)/OD(280nm)</p></td>
+
     <td width="121" valign="top"><p>OD(260/280nm)</p></td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>

Revision as of 01:56, 19 September 2015

Week 12

08/17 - 08/21


NB-Esterase Assay:

MiniPrep 808030 in the plasmid pDG011.


pNP-Assay:

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

Gibson Assembly:

OD measurement of Cluster 4:

 

Concentration (ng/µL)

OD(230nm)

OD(260nm)

OD(280nm)

OD(260/230nm)

OD(260/280nm)

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 migration of the PCR product of prom/pNB Est and Cluster_1_start.

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

 

 

 

 

 

 

 

 

 

 



DNA concentration assay :

 

Concentration (ng/µL)

OD(230nm)

OD(260nm)

OD(280nm)

OD(260/280nm)

OD(260/230nm)

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


Gibson Assembly:

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

Cluster 4A Gibson assembly:

  • Gibson Assembly
  • PCR amplification of cluster 4A Assembly.
  • 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


PCR amplification of Biobricks K808007, K808030, K936011, K936023, K1392932, K316003

  • PCR amplification using Phusion polymerase
  • Gel Migration on a 1% Agarose Gel

The PCR worked for most of our Biobricks with the exception of the biobrick K808030.


DNA concentration measurement

1/25 dilution

DNA concentration (ng/µL)

OD(230nm)

OD(260nm)

OD(280nm)

OD(340nm)

OD(260/280nm)

OD(260/230nm)

808007

4,3

0,08

0,085

0,052

0,006

1,64

1,06

808030

4,6

0,151

0,092

0,058

0,003

1,59

1,61

936011

5,9

0,141

0,118

0,073

0,009

1,61

0,83

936023

5,2

0,097

0,105

0,062

0,003

1,7

1,08

1392932

12,6

0,493

0,251

0,259

0,035

0,97

0,51

316003

5,5

0,1

0,11

0,062

0,0

1,76

1,10


PCR amplification of pSB1C3

  • PCR amplification using Phusion Polymerase.
  • Gel Migration on a 1% agarose gel

Well Number

1

2

3

4

5

6

7

Contains

DNA ladder

Non digested Plasmid

Digested plasmid

No primers

Primer for only

Primer rev only

Both primers


PCR amplification of pSB1C3

  • PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.

 

No primers

Primer for

Primer rev

 

DNAse, RNAse free water

41,75

40,75

40,75

39,75

10X Ex Taq Buffer

5

5

5

5

dNTP

2

2

2

2

rescue_for

0

1

0

1

rescue_rev

0

0

1

1

DNA  Template

1

1

1

1

Ex Taq Polymerase

0,25

0,25

0,25

0,25

Total

50

50

50

50


Cycles:

  • 95°C for 4min
  • 25 cycles :
  • 95°C for 40s
  • annealing for 30s:
  • 60°C: Tube 1-4
  • 61,1°C: Tube 5-8
  • 63°C: Tube 9-12
  • 65,6°C: Tube 13-16
  • 69,2°C: Tube 17-20
  • 72,1°C: Tube 21-24
  • 73,9°C: Tube 25-28
  • 75°C: Tube 29-32
  • 72°C for 80s
  • 72°C for 7min.


  • PCR amplification using Takara Ex Taq

    • Gel Migration on a 1% Agarose Gel.

    PCR amplification of Biobricks K808030

    • PCR Amplification using Takara Ex Taq Polymerase.
    • Gel migration on 1%

    Gel Plan:

    Well Number

    1

    2

    3

    4

    5

     

    DNA ladder

    No primers

    Primer For

    Primer Rev

    Extract



    PCR amplification of pSB1C3

    • PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.
    • Gel Migration on a 1% Agarose Gel.

    PCR amplification of pSB1C3

    • PCR amplification using Takara Ex Taq Polymerase testing 8 annealing temperatures.

     

    No primers

    Primer for

    Primer rev

     

    DNAse, RNAse free water

    41,75

    40,75

    40,75

    39,75

    10X Ex Taq Buffer

    5

    5

    5

    5

    dNTP

    2

    2

    2

    2

    rescue_for

    0

    1

    0

    1

    rescue_rev

    0

    0

    1

    1

    DNA Template

    1

    1

    1

    1

    Ex Taq Polymerase

    0,25

    0,25

    0,25

    0,25

    Total

    50

    50

    50

    50


    Gel Migration on a 1% Agarose Gel

    Enzymatic digest of BBa_K808030

    • Enzymatic digest by XbaI and Spe I of BBa_K808030
    • Gel migration.

    Yeast Assembly

    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(260/280nm)

    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 » : 5µL 10X buffer cutsmart + 0,5µL XbaI + 0,5µL SpeI + 38µL H2O +5µL ADN, digestion at 37°C during 1 hour.
    • tube 2 « Midiprep XbaI/SpeI » : 5µL 10X buffer cut smart + 0,5µL XbaI + 0,5µL SpeI + 38µL H2O + 5µL ADN, digestion at 37°C during 1 hour
    • tube 3 « Miniprep NotI » : 5µL 10X buffer cutsmart + 0,5µL NotI + 38,5µL H2O + 5µL ADN, digestion at 37°C during 1 hour
    • tube 4 « MiDiprep NotI » : 5µL 10X buffer cutsmart + 0,5µL NotI + 38,5µL H2O + 5µL ADN, digestion at 37°C during 1 hour
    • tube 5 « Miniprep CTRL » as negative control : 5µL ADN + 40µL H2O + 5µL 10X buffer cutsmart.
    • tube 6 « Midiprep CTRL » as negative control : 5µL ADN + 40µL H2O + 5µL 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
    • Culture of the plasmid pRS415 in SC_Leu at 30°C at 150rpm for about 72h.
    • PCR Amplification using the Phusion Polymerase of pRS415.
    • Gel Extraction of the PCR products. 


    TPA solubility :

    • Determination of the solubilization products of TPA.
    • Detection of 2-hydroxy-terephthalate using a TECAN spectrometer.
    • Precipitaion of terephthalic acid in H2SO4.


    ^
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