Difference between revisions of "Team:Paris Saclay/Notebook/September/27"

(Created page with "{{Team:Paris_Saclay/notebook_header}} = Tuesday 27<sup>th</sup> September= ==Lab work== ===Visualization=== ====PCR of extracted GFP 1.9 in pSB1C3 (pPS16_020)==== ''By Maxen...")
 
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= Tuesday 27<sup>th</sup> September=
 
= Tuesday 27<sup>th</sup> September=
−
==Lab work==
+
 
 
===Visualization===
 
===Visualization===
  
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''By Maxence & Mahnaz''
 
''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.  
+
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 sent for sequencing the 14th September (clones 2, 7, 8 and 12) and plasmids extracted the 23rd Sepembter (clones 3, 4, 7 and 8) were used.
−
 
+
  
 
For each 50μl of reaction, mix the following reagents :
 
For each 50μl of reaction, mix the following reagents :
Line 50: Line 49:
 
|}
 
|}
  
−
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
+
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
  
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
 
!Matrix
 
!Matrix
−
!Extracted plasmid pSB1C3 containing GFP 1.9 (pPS16_020) from clones 3, 4, 7 & 8
+
!Extracted plasmid pSB1C3 containing GFP 1.9 (pPS16_020)
 
|-
 
|-
 
|Primers
 
|Primers
Line 66: Line 65:
 
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.
 
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.
  
−
GEL
+
PCR products expected were :
 +
 
 +
{| class="wikitable"
 +
|-
 +
!PCR products
 +
!Expected band size (bp)
 +
|-
 +
|GFP 1.9
 +
|1135
 +
|-
 +
|}
 +
 
 +
[[File:T--Paris Saclay--Gel1111113.png|400px|thumb|center|Result of the migration]]
 +
 
 +
====Plasmids extraction of clones 3, 4, 7 and 8 containing GFP 1.9 in pSB1C3 (pPS16_020)====
 +
"By Maxence & Mahnaz"
 +
 
 +
As th quantity of DNA obtained previously was not enough for sequencing, plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit:
 +
*pPS16_020 (GFP 1.9) clone 3
 +
*pPS16_020 (GFP 1.9) clone 4
 +
*pPS16_020 (GFP 1.9) clone 7
 +
*pPS16_020 (GFP 1.9) clone 8
 +
 
 +
====Colony PCR of 23 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_018)====
 +
''By Maxence & Mahnaz''
 +
 
 +
As the two clones selected yesterday were not grown, a new colony PCR was done for 23 clones (16 clones obtained by Gibson with 2 fragments and 7 clones obtained by Gibson with 3 fragments) from the 23rd September. For that purpose, 23 clones were screened and used for the usual [[Team:Paris_Saclay/Experiments##Polymerase_chain_reaction|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:
 +
{| class="wikitable"
 +
|-
 +
!Step
 +
!Temperature
 +
!Time
 +
|-
 +
|Initial denaturation
 +
|95°C
 +
|3 min
 +
|-
 +
|rowspan="3"|30 cycles
 +
|95°C
 +
|30 sec
 +
|-
 +
|48.4°C
 +
|30 sec
 +
|-
 +
|72°C
 +
|30sec
 +
|-
 +
|Final Extension
 +
|72°C
 +
|7 min
 +
|-
 +
|Hold
 +
|4°C
 +
|$\infty$
 +
|}
 +
 
 +
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
 +
 
 +
{| class="wikitable"
 +
|-
 +
!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 :
 +
 
 +
{| class="wikitable"
 +
|-
 +
!PCR products
 +
!Expected band size (bp)
 +
|-
 +
|FKBP - GFP 10 in pSB1C3 (pPS16_019)
 +
|1030
 +
|}
 +
 
 +
[[File:T--Paris Saclay--Gel1111114.png|400px|thumb|center|Result of the migration]]
 +
 
 +
[[File:T--Paris Saclay--Gel1111115.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

Tuesday 27th September

Visualization

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 sent for sequencing the 14th September (clones 2, 7, 8 and 12) and plasmids extracted the 23rd 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
Result of the migration

Plasmids extraction of clones 3, 4, 7 and 8 containing GFP 1.9 in pSB1C3 (pPS16_020)

"By Maxence & Mahnaz"

As th quantity of DNA obtained previously was not enough for sequencing, plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:

  • pPS16_020 (GFP 1.9) clone 3
  • pPS16_020 (GFP 1.9) clone 4
  • pPS16_020 (GFP 1.9) clone 7
  • pPS16_020 (GFP 1.9) clone 8

Colony PCR of 23 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_018)

By Maxence & Mahnaz

As the two clones selected yesterday were not grown, a new colony PCR was done for 23 clones (16 clones obtained by Gibson with 2 fragments and 7 clones obtained by Gibson with 3 fragments) from the 23rd September. For that purpose, 23 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
Result of the migration





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