Difference between revisions of "Team:Paris Saclay/Notebook/September/1"

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===Visualization===
 
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[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
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[[File:T--Paris_saclay--0109.extension.png|400px|thumb|center|Result of migration ]]
 
[[File:T--Paris_saclay--0109.extension.png|400px|thumb|center|Result of migration ]]
 
The result shows that Gibson assembly did not work out.
 
The result shows that Gibson assembly did not work out.
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Latest revision as of 17:01, 9 October 2016

Thursday 1st September

Visualization

Colony PCR of 8 clones containing fragement 1 and 2 (dCas9 Nm GFP 10) in pSB1C3

Mahnaz

Transformed cells on 30th august which had been grown for 2 days were used for colony PCR. For that purpose, 6 clones containing dCas9 NM - GFP 10 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 8 min
30 cycles 95°C 30 sec
Tm 30 sec
72°C t
Final Extension 72°C 10 min
Hold 4°C infinity

Primers used were:

Matrix Clones containing dCas9 NM - GFP 10 in pSB1C3
Primers iPS83 and iPS84
Tm 60.2°C
t 4 min

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 NM - GFP 10 - pSB1C3 3688


Result of migration

The result shows that Gibson assembly did not work out.






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