Difference between revisions of "Team:Paris Saclay/Notebook/September/26"

(Created page with "{{Team:Paris_Saclay/notebook_header}} = Monday 26<sup>th</sup> September= ==Lab work== ===Visualization=== ====Colony PCR of 16 clones containing FKBP - GFP 10 in pSB1C3 (pP...")
 
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= Monday 26<sup>th</sup> September=
 
= Monday 26<sup>th</sup> September=
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==Lab work==
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===Visualization===
 
===Visualization===
  
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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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|FKBP - GFP 10 in pSB1C3 (pPS16_019)
 
|FKBP - GFP 10 in pSB1C3 (pPS16_019)
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|757
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|1030
 
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|}
  
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[[File:T--Paris Saclay--Gel1111111.png|400px|thumb|center|Result of the migration]]
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Two PCR products were at the good size: clones 3 & 11 were selected for sequencing.
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====NanoDrop Measurements====
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''By Maxence, Manhaz & Caroline''
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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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|GFP 1.9 in pSB1C3 (pPS16_020) clone 3<div id="GFP 1.9 in pSB1C3 (pPS16_020) clone 3"></div>
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|8
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|-
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|GFP 1.9 in pSB1C3 (pPS16_020) clone 4<div id="GFP 1.9 in pSB1C3 (pPS16_020) clone 4"></div>
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|14.5
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|-
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|GFP 1.9 in pSB1C3 (pPS16_020) clone 7<div id="GFP 1.9 in pSB1C3 (pPS16_020) clone 7"></div>
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|21.5
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|-
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|GFP 1.9 in pSB1C3 (pPS16_020) clone 8<div id="GFP 1.9 in pSB1C3 (pPS16_020) clone 8"></div>
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|13
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|-
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|PCR product containing FKBP - GFP 10 from the 22nd September<div id="PCR product containing FKBP - GFP 10 from the 22nd"></div>
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|38.19
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|-
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|Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 2<div id="Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 2"></div>
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|235
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|-
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|Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 7<div id="Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 7"></div>
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|51
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|-
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|Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 8<div id="Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 8"></div>
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|200
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|-
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|}
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====Samples preparation for sequencing====
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'By Maxence, Mahnaz & Caroline'
 +
 +
20 µL of plasmids  dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) (clones 2, 7 and 8) were sent to be sequenced. 20 µL of the primers iPS168 (5µM) and iPS169 (5µM) were sent for sequencing.
 +
 +
====Gel of extracted GFP 1.9 in pSB1C3 (pPS16_020)====
 +
''By Maxence, Manhaz & Caroline''
 +
 +
4 µL of extracted plasmids and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity.
 +
 +
[[File:T--Paris Saclay--Gel1111112.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

Monday 26th September

Visualization

Colony PCR of 16 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_018)

By Maxence, Mahnaz & Coline

A colony PCR was done for 16 clones (8 clones obtained by Gibson with 2 fragments and 8 clones obtained by Gibson with 3 fragments) from the 23rd 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
30 cycles 95°C 30 sec
48.4°C 30 sec
72°C 30sec
Final Extension 72°C 7 min
Hold 4°C $\infty$

Primers used were:

Matrix Clones containing FKBP - GFP 10 in pSB1C3 (pPS16_19)
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)
FKBP - GFP 10 in pSB1C3 (pPS16_019) 1030
Result of the migration

Two PCR products were at the good size: clones 3 & 11 were selected for sequencing.

NanoDrop Measurements

By Maxence, Manhaz & Caroline

Sample Concentration (ng/µL)
GFP 1.9 in pSB1C3 (pPS16_020) clone 3
 8
GFP 1.9 in pSB1C3 (pPS16_020) clone 4
 14.5
GFP 1.9 in pSB1C3 (pPS16_020) clone 7
 21.5
GFP 1.9 in pSB1C3 (pPS16_020) clone 8
 13
PCR product containing FKBP - GFP 10 from the 22nd September
 38.19
Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 2
 235
Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 7
 51
Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 8
 200

Samples preparation for sequencing

'By Maxence, Mahnaz & Caroline'

20 µL of plasmids dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) (clones 2, 7 and 8) were sent to be sequenced. 20 µL of the primers iPS168 (5µM) and iPS169 (5µM) were sent for sequencing.

Gel of extracted GFP 1.9 in pSB1C3 (pPS16_020)

By Maxence, Manhaz & Caroline

4 µL of extracted plasmids and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity.

Result of the migration





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