Difference between revisions of "Team:Paris Saclay/Notebook/September/21"

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= Wednesday 21<sup>st</sup> September=
 
= Wednesday 21<sup>st</sup> September=
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==Lab work==
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===Visualization===
 
===Visualization===
  
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====Colony PCR of 16 clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)====
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====Colony PCR of 16 clones containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)====
 
''By Maxence, Mahnaz & Coline''
 
''By Maxence, Mahnaz & Coline''
  
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|3 min
 
|3 min
 
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|rowspan="3"|30 cycles
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|rowspan="3"|25 cycles
 
|95°C
 
|95°C
 
|30 sec
 
|30 sec
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|72°C
 
|72°C
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|1min 30sec
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|4min
 
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|Final Extension
 
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[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
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[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
  
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
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!Matrix
 
!Matrix
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!Clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
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!Clones containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)
 
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|Primers
 
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!Expected band size (bp)
 
!Expected band size (bp)
 
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|FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
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|dCas9 ST - GFP 11
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|1500
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|3800
 
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[[File:T--Paris Saclay--Gel111111.png|400px|thumb|center|Result of the migration]]
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 17:09, 9 October 2016

Wednesday 21st September

Visualization

Colony PCR of 16 clones containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)

By Maxence, Mahnaz & Coline

A colony PCR was done for 16 clones from the 20th September. For that purpose, 16 clones were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.

For each clones contained in 20 μl water, 5.13 μL of the following mix were added :

  • 2.5 µL DreamTaq Buffer
  • 0.5 µL of dNTPs (10mM)
  • 1 µL of each primer mix (10µM)
  • 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step Temperature Time
Initial denaturation 95°C 3 min
25 cycles 95°C 30 sec
48.4°C 30 sec
72°C 4min
Final Extension 72°C 7 min
Hold 4°C $\infty$

Primers used were:

Matrix Clones containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)
Primers iPS168 and iPS169

After amplification, 3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

PCR products Expected band size (bp)
dCas9 ST - GFP 11 3800
Result of the migration





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