Difference between revisions of "Team:Paris Saclay/Notebook/September/28"

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= Wednesday 28<sup>th</sup> September=
 
= Wednesday 28<sup>th</sup> September=
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==Lab work==
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===Visualization===
 
===Visualization===
  
 
==== Glycerol stocks of 8 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_018)====
 
==== Glycerol stocks of 8 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_018)====
−
"By Maxence & Mahnaz"
+
''By Maxence & Mahnaz''
  
 
The glycerol stock of the bacteria with the following plasmids were made.
 
The glycerol stock of the bacteria with the following plasmids were made.
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*pPS16_018 (FKBP - GFP 10) clone 5 (Gibson 3 fragments)
 
*pPS16_018 (FKBP - GFP 10) clone 5 (Gibson 3 fragments)
 
*pPS16_018 (FKBP - GFP 10) clone 6 (Gibson 3 fragments)
 
*pPS16_018 (FKBP - GFP 10) clone 6 (Gibson 3 fragments)
 +
 +
====NanoDrop Measurements====
 +
''By Maxence & Manhaz''
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 +
{| class="wikitable"
 +
!Sample
 +
!Concentration (ng/µL)
 +
|-
 +
|FKBP - GFP 10 clone 4 (Gibson 2 fragments)<div id="FKBP - GFP 10 clone 4 (Gibson 2 fragments)"></div>
 +
|31.05
 +
|-
 +
|FKBP - GFP 10 clone 7 (Gibson 2 fragments)<div id="FKBP - GFP 10 clone 7 (Gibson 2 fragments)"></div>
 +
|271.52
 +
|-
 +
|FKBP - GFP 10 clone 10 (Gibson 2 fragments)<div id="FKBP - GFP 10 clone 10 (Gibson 2 fragments)"></div>
 +
|62.18
 +
|-
 +
|FKBP - GFP 10 clone 11 (Gibson 2 fragments)<div id="FKBP - GFP 10 clone 11 (Gibson 2 fragments)"></div>
 +
|244.56
 +
|-
 +
|FKBP - GFP 10 clone 2 (Gibson 3 fragments)<div id="FKBP - GFP 10 clone 2 (Gibson 3 fragments)"></div>
 +
|171.08
 +
|-
 +
|FKBP - GFP 10 clone 4 (Gibson 3 fragments)<div id="FKBP - GFP 10 clone 4 (Gibson 3 fragments)"></div>
 +
|420.69
 +
|-
 +
|FKBP - GFP 10 clone 5 (Gibson 3 fragments)<div id="FKBP - GFP 10 clone 5 (Gibson 3 fragments)"></div>
 +
|88.42
 +
|-
 +
|FKBP - GFP 10 clone 6 (Gibson 3 fragments)<div id="FKBP - GFP 10 clone 6 (Gibson 6 fragments)"></div>
 +
|43.97
 +
|-
 +
|}
  
 
====PCR of extracted FKBP - GFP 10 in pSB1C3 (pPS16_018)====
 
====PCR of extracted FKBP - GFP 10 in pSB1C3 (pPS16_018)====
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|}
 
|}
  
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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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{| class="wikitable"
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|}
 
|}
  
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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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|}
 
|}
  
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[[File:T--Paris Saclay--Gel11111111.png|400px|thumb|center|Result of the migration]]
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[[File:T--Paris Saclay--Gel11111112.png|400px|thumb|center|Result of the migration]]
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 15:51, 14 October 2016

Wednesday 28th September

Visualization

Glycerol stocks of 8 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_018)

By Maxence & Mahnaz

The glycerol stock of the bacteria with the following plasmids were made.

  • pPS16_018 (FKBP - GFP 10) clone 4 (Gibson 2 fragments)
  • pPS16_018 (FKBP - GFP 10) clone 7 (Gibson 2 fragments)
  • pPS16_018 (FKBP - GFP 10) clone 10 (Gibson 2 fragments)
  • pPS16_018 (FKBP - GFP 10) clone 11 (Gibson 2 fragments)
  • pPS16_018 (FKBP - GFP 10) clone 2 (Gibson 3 fragments)
  • pPS16_018 (FKBP - GFP 10) clone 4 (Gibson 3 fragments)
  • pPS16_018 (FKBP - GFP 10) clone 5 (Gibson 3 fragments)
  • pPS16_018 (FKBP - GFP 10) clone 6 (Gibson 3 fragments)

500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.

Plasmids extraction of 8 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_018)

"By Maxence & Mahnaz"

The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:

  • pPS16_018 (FKBP - GFP 10) clone 4 (Gibson 2 fragments)
  • pPS16_018 (FKBP - GFP 10) clone 7 (Gibson 2 fragments)
  • pPS16_018 (FKBP - GFP 10) clone 10 (Gibson 2 fragments)
  • pPS16_018 (FKBP - GFP 10) clone 11 (Gibson 2 fragments)
  • pPS16_018 (FKBP - GFP 10) clone 2 (Gibson 3 fragments)
  • pPS16_018 (FKBP - GFP 10) clone 4 (Gibson 3 fragments)
  • pPS16_018 (FKBP - GFP 10) clone 5 (Gibson 3 fragments)
  • pPS16_018 (FKBP - GFP 10) clone 6 (Gibson 3 fragments)

NanoDrop Measurements

By Maxence & Manhaz

Sample Concentration (ng/µL)
FKBP - GFP 10 clone 4 (Gibson 2 fragments)
 31.05
FKBP - GFP 10 clone 7 (Gibson 2 fragments)
 271.52
FKBP - GFP 10 clone 10 (Gibson 2 fragments)
 62.18
FKBP - GFP 10 clone 11 (Gibson 2 fragments)
 244.56
FKBP - GFP 10 clone 2 (Gibson 3 fragments)
 171.08
FKBP - GFP 10 clone 4 (Gibson 3 fragments)
 420.69
FKBP - GFP 10 clone 5 (Gibson 3 fragments)
 88.42
FKBP - GFP 10 clone 6 (Gibson 3 fragments)
 43.97

PCR of extracted FKBP - GFP 10 in pSB1C3 (pPS16_018)

By Maxence & Mahnaz

In order to verify if extracted plasmids contain FKBP - GFP 10, a PCR was run with iPS168 & iPS169 to amplify the insert. For that purpose, plasmids extracted today (clones 4, 7, 10 & 11 for 2 fragments Gibson and clones 2, 4, 4 & 6 for 3 fragments Gibson) were used.

For each 50μl of reaction, mix the following reagents :

  • 1 µL of matrix
  • 1 µL of dNTPs (10mM)
  • 2.5 µL of each primer mix (10µM)
  • 10 µL of buffer (5X)
  • 0,5 µL of Phusion polymerase
  • 32.5 µL of nuclease free water

Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow:

Step Temperature Time
Initial denaturation 95°C 30sec
30 cycles 95°C 30sec
64.4°C 30sec
72°C 30sec
Final Extension 72°C 5min
Hold 4°C $\infty$

Primers used were:

Matrix Extracted plasmid pSB1C3 containing GFP 1.9 (pPS16_020)
Primers iPS168 and iPS169

PCR of extracted GFP 1.9 in pSB1C3 (pPS16_020)

By Maxence & Mahnaz

In order to verify if extracted plasmids contain GFP 1.9, a PCR was run with iPS168 & iPS169 to amplify the insert. For that purpose, plasmids extracted the 27th Sepembter (clones 3, 4, 7 and 8) were used.

For each 50μl of reaction, mix the following reagents :

  • 1 µL of matrix
  • 1 µL of dNTPs (10mM)
  • 2.5 µL of each primer mix (10µM)
  • 10 µL of buffer (5X)
  • 0,5 µL of Phusion polymerase
  • 32.5 µL of nuclease free water

Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow:

Step Temperature Time
Initial denaturation 95°C 30sec
30 cycles 95°C 30sec
64.4°C 30sec
72°C 30sec
Final Extension 72°C 5min
Hold 4°C $\infty$

Primers used were:

Matrix Extracted plasmid pSB1C3 containing GFP 1.9 (pPS16_020)
Primers iPS168 and iPS169

Gel of PCR products

By Maxence & Manhaz

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

PCR products expected were :

PCR products Expected band size (bp)
GFP 1.9 1135
FKBP - GFP 10 1030
Result of the migration
Result of the migration





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