Difference between revisions of "Team:Paris Saclay/Notebook/October/5"

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=Wednesday 5<sup>th</sup> October=
 
=Wednesday 5<sup>th</sup> October=
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==Lab work==
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===Visualization===
 
===Visualization===
 +
 +
====Colony PCR of 13 clones containing GFP 1.9 in pSB1C3 (pPS16_020)====
 +
''By Maxence & Yacine''
 +
 +
For that purpose, 13 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:
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{| class="wikitable"
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|-
 +
!Step
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!Temperature
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!Time
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|-
 +
|Initial denaturation
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|95°C
 +
|3 min
 +
|-
 +
|rowspan="3"|30 cycles
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|95°C
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|30 sec
 +
|-
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|48.4°C
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|30 sec
 +
|-
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|72°C
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|30 sec
 +
|-
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|Final Extension
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|72°C
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|7 min
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|-
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|Hold
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|4°C
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|$\infty$
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|}
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 +
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
 +
 +
{| class="wikitable"
 +
|-
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!Matrix
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!Clones containing GFP 1.9 in pSB1C3 (pPS16_020)
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|-
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|Primers
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|iPS168 and iPS169
 +
|-
 +
|}
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 +
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
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!Expected band size (bp)
 +
|-
 +
|GFP 1.9
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|1135
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|}
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 +
No PCR products were obtained.
  
 
====Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6====
 
====Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6====
−
"By Maxence & Yacine"
+
''By Maxence & Yacine''
  
−
The following plasmids were extracted using a [[Team:Paris_Saclay/Experiments#Plasmid_DNA_extraction|"standard Plasmid Miniprep"]] kit:
+
The following plasmids were extracted using a [[Team:Paris_Saclay/Experiments#Plasmid_DNA_extraction|"standard Plasmid Miniprep"]]:
 
*pPS16_018 (FKBP - GFP 10) clone 6
 
*pPS16_018 (FKBP - GFP 10) clone 6
 
*pPS16_019 (FRB - GFP 11) clone 4
 
*pPS16_019 (FRB - GFP 11) clone 4
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And FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6 was cut by restriction enzymes PstI & XbaI and was treated by the alkaline phosphatase FastAP (dephosphorylation of cloning vector DNA to prevent recircularization during ligation) as following:
 
And FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6 was cut by restriction enzymes PstI & XbaI and was treated by the alkaline phosphatase FastAP (dephosphorylation of cloning vector DNA to prevent recircularization during ligation) as following:
−
 
  
 
* 20 µL of FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6
 
* 20 µL of FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6
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|}
 
|}
  
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GEL
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[[File:T--Paris Saclay--Gel111111112.png|400px|thumb|center|Result of the migration]]
  
 
The digestion products were cleaned up from the gel by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]].
 
The digestion products were cleaned up from the gel by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]].

Latest revision as of 15:54, 14 October 2016

Wednesday 5th October

Visualization

Colony PCR of 13 clones containing GFP 1.9 in pSB1C3 (pPS16_020)

By Maxence & Yacine

For that purpose, 13 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 30 sec
Final Extension 72°C 7 min
Hold 4°C $\infty$

Primers used were:

Matrix Clones containing GFP 1.9 in pSB1C3 (pPS16_020)
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)
GFP 1.9 1135

No PCR products were obtained.

Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6

By Maxence & Yacine

The following plasmids were extracted using a "standard Plasmid Miniprep":

  • pPS16_018 (FKBP - GFP 10) clone 6
  • pPS16_019 (FRB - GFP 11) clone 4

Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation

By Maxence & Yacine

For that purpose, FRB - GFP 11 (pPS16_019) clone 4 was cut by restriction enzymes XbaI & PstI as following:

  • 20 µL of FRB - GFP 11 (pPS16_019) clone 4
  • 4 µL of buffer FD
  • 2 µL of restriction enzyme XbaI
  • 2 µL of restriction enzyme PstI
  • 10 µL of water

And FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6 was cut by restriction enzymes PstI & XbaI and was treated by the alkaline phosphatase FastAP (dephosphorylation of cloning vector DNA to prevent recircularization during ligation) as following:

  • 20 µL of FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6
  • 4 µL of buffer FD
  • 2 µL of restriction enzyme SpeI
  • 2 µL of restriction enzyme PstI
  • 2 µL of alkaline phosphatase FastAP
  • 10 µL of water

The mix were incubated for 20 minutes at 37°C. Then, 40 µL of each digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

Migration products expected were :

Migration products Expected band size (bp)
Template digested (pPS16_019 treated by XbaI & PstI) 727
Vector digested (pPS16_018 treated by SpeI & PstI) 2784
Result of the migration

The digestion products were cleaned up from the gel by using the NucleoSpin Gel and PCR Clean-up kit protocol.






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