Difference between revisions of "Team:Paris Saclay/Notebook/September/16"

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= Friday 16<sup>th</sup> September=
 
= Friday 16<sup>th</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 in pSB1C3====
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====Colony PCR of 16 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_019)====
 
''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:
  
 
{| class="wikitable"
 
{| class="wikitable"
 
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!Matrix
 
!Matrix
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!Clones containing FKBP - GFP 10 in pSB1C3
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!Clones containing FKBP - GFP 10 in pSB1C3 (pPS16_19)
 
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|Primers
 
|Primers
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!Expected band size (bp)
 
!Expected band size (bp)
 
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|FKBP - GFP 10 in pSB1C3
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|FKBP - GFP 10 in pSB1C3 (pPS16_019)
 
|757
 
|757
 
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GEL
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[[File:T--Paris Saclay--Gel1118.png|400px|thumb|center|Result of the migration]]
  
 
Some PCR products were at the good size. Clones 7, 8, 9 and 10 were selected and were grown at 37°C overnight.
 
Some PCR products were at the good size. Clones 7, 8, 9 and 10 were selected and were grown at 37°C overnight.
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 17:06, 9 October 2016

Friday 16th September

Visualization

Colony PCR of 16 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_019)

By Mahnaz

Colonies were obtained for the Gibson done the 15th September. A colony PCR was done for 16 clones from the 15th September. For that purpose, 16 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
30 cycles 95°C 30 sec
61°C 30 sec
72°C 30 sec
Final Extension 72°C 7 min
Hold 4°C $\infinity\$

Primers used were:

Matrix Clones containing FKBP - GFP 10 in pSB1C3 (pPS16_19)
Primers iPS83 and iPS84

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)
FKBP - GFP 10 in pSB1C3 (pPS16_019) 757
Result of the migration

Some PCR products were at the good size. Clones 7, 8, 9 and 10 were selected and were grown at 37°C overnight.





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