Difference between revisions of "Team:Paris Saclay/Notebook/August/25"

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(→‎pPS16_014 (SgRNA Nm) clone 5, Gibson Assembly's Product (Fragment3-4) clone 6, and pPS16_009 clone 4 extraction)
 
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{{Team:Paris_Saclay/notebook_header}}
 
{{Team:Paris_Saclay/notebook_header}}
−
= Thursday 25<sup>th</sup> August=
+
 
−
==Lab work==
+
=Thursday 25<sup>th</sup> August=
 +
 
 
===Visualization===
 
===Visualization===
−
====Extraction of plasmids containing sgRNA Nm, fragment 3 + 4 (from the HIfi assembly mix) , GFP1-9====
+
 
 +
====pPS16_014 (SgRNA Nm) clone 5, Gibson Assembly's Product (Fragment3-4) clone 6, and pPS16_009 clone 4 extraction====
 
''By Terrence''
 
''By Terrence''
  
−
The extraction was carried out following the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|usual protocol]].
+
The extraction was carried out following the [[Team:Paris_Saclay/Experiments#DNA_extraction_using_the_Invitrogen_ChargeSwitch.C2.AE-Pro_Plasmid_Miniprep_Kit|usual protocol]]
  
−
The plasmid PSB1C3  with the insert :
+
The Gibson Assembly's Product (Fragment3-4) clone 6 and pPS16_009 clone 4 extraction products were digested with the restriction enzymes XBaI and PSTI
−
- SgRNA Nm were extracted from the  clone 5
+
−
- Fragment 3 + 4 were extracted from the  clone 6
+
−
- GFP1-9 were extracted from the clone 4
+
−
+
  
 +
====Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB====
 +
''By Mahnaz''
  
 +
Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 12 clones containing sg-Nm and 12 clones containing FRB 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 + 50 µg/mL Amp and liquid cultures (LB + 50 µg/mL Amp) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.
  
−
====Digestion of PSB1C3 containing the fragment 3 + 4 assembly and PSB1C3 with GFP1-9's insert.====
+
For each clones contained in 20 μl water, 4.13 μL of the following mix were added :
−
''By Terrence''
+
* 2.5 µL DreamTaq Buffer
 +
* 0.5 µL of dNTPs (10mM)
 +
* 0.5 µL of each primer(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"|25 cycles
 +
|94°C
 +
|30 sec
 +
|-
 +
|Tm
 +
|30 sec
 +
|-
 +
|72°C
 +
|t
 +
|-
 +
|Final Extension
 +
|72°C
 +
|5min
 +
|-
 +
|Hold
 +
|4°C
 +
|$\infinity\$
 +
|}
 +
 
 +
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
 +
 
 +
{| class="wikitable"
 +
|-
 +
!Matrix
 +
!plasmid pJET coding sg-Nm
 +
!plasmid pJET coding FRB
 +
|-
 +
|Primers
 +
|pJET R and pJET F
 +
|pJET R and pJET F
 +
|-
 +
|Tm
 +
|60.0C
 +
|60.0C
 +
|-
 +
|t
 +
|15 sec
 +
|15 sec
 +
|}
 +
 
 +
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)
 +
|-
 +
|FRB
 +
|374
 +
|-
 +
|sg-Nm
 +
|362
 +
|}
 +
 
 +
[[File:T--Paris_Saclay--20160825_PCR_gel_Nm_Frb.jpg|400px|thumb|right|We can't see anything, the only visibles strands are the priers sequence, The result is not satisfying]]
  
−
Extracted plasmids were digest with XbaI and PstI, following [[Team:Paris_Saclay/Experiments#PlasmidDigestion|usual protocol]].
+
====Q5 high fidelity PCR of ligation products pPS16_001-pPS16_002 and pPS16_003-pPS16_004====
 +
''By mathilde''
  
 +
Fragment 1 (pPS16_001-pPS16_002) and fragment 2 (pPS16_003-pPS16_004E) were each tested three times (A,B,C) according to the following protocol :
  
 +
* 10µL of Q5 buffer
 +
* 1µL of dNTPs (10mM)
 +
* Primers mix (1µL each)
 +
* 1µL of ligation product
 +
* 0,25 µL of Q5 high fidelity polymerase
 +
* 37,75 µL of nuclease free water
  
 +
The primers used were IPS83 and IPS 122 for the fragment 1, and IPS 84 and 123 for the fragment 2.
 +
The PCR programm featured 30s of initial denaturation, 72°c of annealing temperature for 30s and 2min of final extension.
  
  
 +
====Gel electrophoresis of Q5 PCR ligation products pPS16_001-pPS16_002 and pPS16_003-pPS16_004====
 +
PCR products were put to migration following the usual protocol.
  
 +
{| class="wikitable"
 +
|-
 +
!PCR products
 +
!Expected band size (bp)
 +
|-
 +
|pPS16_001-pPS16_002
 +
|1920
 +
|-
 +
|pPS16_003-pPS16_004
 +
|1831
 +
|}
  
 +
[[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]]
 +
Only pPS16_001-pPS16_002 ligation product shows strands at the expected size.
  
 +
====Ligation of pPS16_003-pPS16_004====
 +
''By Mathilde''
  
 +
* 4 µL of pPS16_003 plasmid
 +
* 4 µL of pPS16_004 plasmid
 +
* 1µL of T4 Buffer 10X
 +
* 1µL of T4 Ligase
 +
The solution is put in incubation at 4°c over night.
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 11:59, 17 October 2016

Thursday 25th August

Visualization

pPS16_014 (SgRNA Nm) clone 5, Gibson Assembly's Product (Fragment3-4) clone 6, and pPS16_009 clone 4 extraction

By Terrence

The extraction was carried out following the usual protocol

The Gibson Assembly's Product (Fragment3-4) clone 6 and pPS16_009 clone 4 extraction products were digested with the restriction enzymes XBaI and PSTI

Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB

By Mahnaz

Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 12 clones containing sg-Nm and 12 clones containing FRB were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 50 µg/mL Amp and liquid cultures (LB + 50 µg/mL Amp) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.

For each clones contained in 20 μl water, 4.13 μL of the following mix were added :

  • 2.5 µL DreamTaq Buffer
  • 0.5 µL of dNTPs (10mM)
  • 0.5 µL of each primer(10µM)
  • 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step Temperature Time
Initial denaturation 95°C 3 min
25 cycles 94°C 30 sec
Tm 30 sec
72°C t
Final Extension 72°C 5min
Hold 4°C $\infinity\$

Primers used were:

Matrix plasmid pJET coding sg-Nm plasmid pJET coding FRB
Primers pJET R and pJET F pJET R and pJET F
Tm 60.0C 60.0C
t 15 sec 15 sec

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)
FRB 374
sg-Nm 362
We can't see anything, the only visibles strands are the priers sequence, The result is not satisfying

Q5 high fidelity PCR of ligation products pPS16_001-pPS16_002 and pPS16_003-pPS16_004

By mathilde

Fragment 1 (pPS16_001-pPS16_002) and fragment 2 (pPS16_003-pPS16_004E) were each tested three times (A,B,C) according to the following protocol :

  • 10µL of Q5 buffer
  • 1µL of dNTPs (10mM)
  • Primers mix (1µL each)
  • 1µL of ligation product
  • 0,25 µL of Q5 high fidelity polymerase
  • 37,75 µL of nuclease free water

The primers used were IPS83 and IPS 122 for the fragment 1, and IPS 84 and 123 for the fragment 2. The PCR programm featured 30s of initial denaturation, 72°c of annealing temperature for 30s and 2min of final extension.


Gel electrophoresis of Q5 PCR ligation products pPS16_001-pPS16_002 and pPS16_003-pPS16_004

PCR products were put to migration following the usual protocol.

PCR products Expected band size (bp)
pPS16_001-pPS16_002 1920
pPS16_003-pPS16_004 1831
Result of the migration

Only pPS16_001-pPS16_002 ligation product shows strands at the expected size.

Ligation of pPS16_003-pPS16_004

By Mathilde

  • 4 µL of pPS16_003 plasmid
  • 4 µL of pPS16_004 plasmid
  • 1µL of T4 Buffer 10X
  • 1µL of T4 Ligase

The solution is put in incubation at 4°c over night.





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