Difference between revisions of "Team:Paris Saclay/Notebook/August/29"

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PCR sur colonie
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{{Team:Paris_Saclay/notebook_header}}
  
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= Monday 29<sup>th</sup> August=
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===Visualization===
 
====PCR of the plasmid extraction from clonies 4,5,9,12 containing FRB sequence in pJET plasmid====
 
====PCR of the plasmid extraction from clonies 4,5,9,12 containing FRB sequence in pJET plasmid====
 
''By Mahnaz''
 
''By Mahnaz''
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[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
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[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
  
 
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|Tm
 
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|60°C
 
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!Expected band size (bp)
 
!Expected band size (bp)
 
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|dCas9 NM - GFP 10 - pSB1C3
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|FRB in pJET
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|3688
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|474
 
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[[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]]
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[[File:T--Paris_saclay--2908.extension.png|400px|thumb|center|Result of migration ]]
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The expected size was 474 pb and the 4 plasmids were sent to sequencing.
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====Samples preparation for sequencing====
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''by Mahnaz''
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15 µL of plasmides plasmids coding FRB from colonies 4,5,9 and 12 were sent to be sequenced. 20 µL of the primers pJET F (5µM) and pJET R (5µM) were sent for sequencing.
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====NanoDrop Measurements====
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''By Mahnaz''
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{| class="wikitable"
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!Sample
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!Concentration (ng/µL)
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|-
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|PCR fragment FRB clone 4<div id="PCR fragment FRB clone 4"></div>
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|58.43
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|-
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|PCR fragment FRB clone 5<div id="PCR fragment FRB clone 5"></div>
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|320.75
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|-
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|PCR fragment FRB clone 9<div id="PCR fragment FRB clone 9"></div>
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|830.99
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|-
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|PCR fragment FRB clone 12<div id="PCR fragment FRB clone 12"></div>
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|473.37
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|-
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|}
  
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GEL GEL GEL
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{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 17:00, 9 October 2016

Monday 29th August

Visualization

PCR of the plasmid extraction from clonies 4,5,9,12 containing FRB sequence in pJET plasmid

By Mahnaz

  • 2.5 µL DreamTaq Buffer
  • 0.5 µL of dNTPs (10mM)
  • 1 µL of each primer (10µM) primer pJET R and F
  • 0.13 μl of DreamTaq Pol
  • 20 µl H2O

PCR was performed as follow:

Step Temperature Time
Initial denaturation 95°C 3 min
30 cycles 95°C 30 sec
52 30 sec
72°C 1 min
Final Extension 72°C 7min
Hold 4°C \infinity\

Primers used were:

Matrix Clones containing dCas9 NM - GFP 10 in pSB1C3
Primers pJET R and F
Tm 60°C
t 1 min 30

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)
FRB in pJET 474
Result of migration

The expected size was 474 pb and the 4 plasmids were sent to sequencing.

Samples preparation for sequencing

by Mahnaz

15 µL of plasmides plasmids coding FRB from colonies 4,5,9 and 12 were sent to be sequenced. 20 µL of the primers pJET F (5µM) and pJET R (5µM) were sent for sequencing.

NanoDrop Measurements

By Mahnaz

Sample Concentration (ng/µL)
PCR fragment FRB clone 4
 58.43
PCR fragment FRB clone 5
 320.75
PCR fragment FRB clone 9
 830.99
PCR fragment FRB clone 12
 473.37





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