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

(→‎Q5 PCR on products of 1.1 and 1.2, and of 2.1 and 2.2 gBlocks ligation)
(→‎pPS16_014 (SgRNA Nm) clone 5, Gibson Assembly's Product (Fragment3-4) clone 6, and pPS16_009 clone 4 extraction)
 
(23 intermediate revisions by 6 users not shown)
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{{Team:Paris_Saclay/notebook_header}}
 
{{Team:Paris_Saclay/notebook_header}}
−
= Thursday 25<sup>th</sup> August=
 
−
==Lab work==
 
−
===Visualization===
 
−
====Extraction of plasmids containing sgRNA Nm, fragment 3 + 4 (from the HIfi assembly mix) , GFP1-9====
 
−
''By Terrence''
 
−
 
−
The extraction was carried out following the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|usual protocol]].
 
−
 
−
The plasmid PSB1C3  with the insert :
 
−
- 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
 
−
 
  
 +
=Thursday 25<sup>th</sup> August=
  
 +
===Visualization===
  
−
====Digestion of PSB1C3 containing the fragment 3 + 4 assembly and PSB1C3 with GFP1-9's insert.====
+
====pPS16_014 (SgRNA Nm) clone 5, Gibson Assembly's Product (Fragment3-4) clone 6, and pPS16_009 clone 4 extraction====
 
''By Terrence''
 
''By Terrence''
  
−
Extracted plasmids were digest with XbaI and PstI, following [[Team:Paris_Saclay/Experiments#PlasmidDigestion|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 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''
  
−
[[File:T--Paris_Saclay--20160825_digestion_gel.jpg|400px|thumb|right|Result of the digestion]]
+
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.
  
 +
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
  
−
====Q5 PCR on products of 1.1 and 1.2, and of 2.1 and 2.2 gBlocks ligation====
+
PCR was performed as follow:  
−
''By Mathilde''
+
−
 
+
−
Q5 PCR was performed directly on gBlocks to amplify them following the protocol:
+
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Prepare enough PCR master mix for two sets of triplicats analyzed plus one extra. For each 50μl of reaction, mix the following reagents :
+
−
* 1µL of ligation product
+
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* 1µ of dNTPs (10mM)
+
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* 1µL of each primer mix (10µM)
+
−
* 10µL of q5 buffer
+
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* 0,25µL of Q5 high fidelity polymerase
+
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* 35,75µL of nuclease free water
+
−
 
+
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*Mix gently and aliquot 50μl of the mix into the PCR tubes on ice.
+
−
*Perform PCR as follow:  
+
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
Line 47: Line 31:
 
|-
 
|-
 
|Initial denaturation
 
|Initial denaturation
−
|98°C
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|95°C
−
|30sec
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|3 min
 
|-
 
|-
−
|rowspan="3"|30 cycles
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|rowspan="3"|25 cycles
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|98°C
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|94°C
−
|5sec
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|30 sec
 
|-
 
|-
−
|72°c
+
|Tm
−
|30sec
+
|30 sec
 
|-
 
|-
 
|72°C
 
|72°C
−
|1min
+
|t
 
|-
 
|-
 
|Final Extension
 
|Final Extension
 
|72°C
 
|72°C
−
|2min
+
|5min
 
|-
 
|-
 
|Hold
 
|Hold
 
|4°C
 
|4°C
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|$\infty$
+
|$\infinity\$
 
|}
 
|}
−
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
+
 
 +
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
  
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
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!gBlocks
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!Matrix
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!1.1 and 1.2 gBlocks ligation
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!plasmid pJET coding sg-Nm
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!2.1 and 2.2 gBlocks ligation
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!plasmid pJET coding FRB
 
|-
 
|-
 
|Primers
 
|Primers
−
|iPS83 and iPS122
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|pJET R and pJET F
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|iPS184 and iPS123
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|pJET R and pJET F
 +
|-
 +
|Tm
 +
|60.0C
 +
|60.0C
 +
|-
 +
|t
 +
|15 sec
 +
|15 sec
 
|}
 
|}
  
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After amplification, 3 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.
+
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 were :
Line 87: Line 80:
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
−
!gBlocks
+
!PCR products
−
!expected band size (bp)
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!Expected band size (bp)
 
|-
 
|-
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|1.1 and 1.2 gBlocks ligation
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|FRB
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|960
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|374
 
|-
 
|-
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|4.1 and 4.2 gBlocks ligation
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|sg-Nm
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|1994
+
|362
−
|}  
+
|}
  
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[[File:T--Paris_Saclay--20160817_PCR_gblocks_ligation.jpeg|500px|thumb|right|Migration of gBlocks and ligation]]
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[[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]]
  
 +
====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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