Difference between revisions of "Team:Paris Saclay/Notebook/August/24"

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= Wednesday 24<sup>th</sup> August=
 
= Wednesday 24<sup>th</sup> August=
−
==Lab work==
+
 
 
===Visualization===
 
===Visualization===
−
====Migration gel of Gibson (fragment 3 + fragment 4) and GFP in PSB1C3====
+
====Gel Electrophoresis of Gibson Assembly's products(fragment 3-4) and GFP 1-9 cloned in PSB1C3====
 
''By Terrence''
 
''By Terrence''
  
 +
[[File:T--Paris_Saclay--20160824_Gibson_frag4-3.jpg|400px|thumb|right|Colony PCR of 12 colonies transformed with Gibson Assembly products (fragment 3-4).]]
 +
 +
[[File:T--Paris_Saclay--20160824_PCR_transfo_GFP_PSB1C3.jpg|400px|thumb|right|Colony PCR of 12 colonies containing the GFP 1-9.]]- Clones 2, 4, 7 and 11 are as expected for GFP 1.9.
 +
 +
====pPS16_002 (1.2) clones 8 and 14 and pPS16_014(SgRna Nm) clone 12 Extraction ====
 +
''By Terrence''
 +
 +
The extraction was carried out following the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|usual protocol]].
 +
 +
 +
[[File:T--Paris_Saclay--20160824_extraction_1-2_Nm.jpg|400px|thumb|right|Result of the extraction]]
 +
 +
 +
 +
Nano drop :
 +
 +
{| class="wikitable"
 +
|-
 +
!
 +
!concentration (ng/µL)
 +
!260/230
 +
!260/280
 +
|-
 +
|1.2.14
 +
|47.81
 +
|1.64
 +
|1.84
 +
|-
 +
|1.2.8
 +
|834.5
 +
|2.40
 +
|1.99
 +
|-
 +
|SgRna Nm 12
 +
|124.21
 +
|2.00
 +
|1.89
 +
|}
 +
 +
====Transformation of gblockFRB in pJET and gblock Sg_RNA_NM in pJET====
 +
''By Terrence''
 +
 +
 +
First we made a ligation with
 +
 +
{| class="wikitable"
 +
|-
 +
!Component
 +
!Volume (µL)
 +
|-
 +
|Reaction Buffer
 +
|10
 +
|-
 +
|PCR Product (FRB or Nm)
 +
|1
 +
|-
 +
|pJET1.2/blunt
 +
|1
 +
|-
 +
|water, nuclease free
 +
|7
 +
|-
 +
|T4 DNA Ligase
 +
|1
 +
|-
 +
|}
 +
 +
Then we transformed 2µL of ligation product into 50 µL of DH5a competent cells, following the [[Team:Paris_Saclay/Experiments#Heat_shock_competent_cells|usual protocol]].
 +
 +
Transformation products were plated with three different volume : 50µL , 150µl and 350µL. 350µL only for the control.
 +
 +
====Plasmid DNA extraction (Midi Prep)====
 +
''By Léa & Manhaz''
 +
 +
We have decided to use the plasmid coding dCas9 Nm to amplify the fragement corresponding g-block 1.2.
 +
200 mL of transformed DH5a cells with Adgene dCas9 Nm plasmids (clone 1 and 2) cultures were extracted usind a Midi Prep kit. It was resuspended into 50µL of sterile water.
 +
 +
The plasmid then reserved in -20.
 +
 +
====PCR Q5 on plasmid ====
 +
''By Léa & Manhaz''
  
−
[[File:T--Paris_Saclay--20160824_Gibson_frag4-3.jpg|400px|thumb|right|Clone 6 and 8 seems correct for the Gibson.]]
+
Plasmid pJet cotnains gblock 1.2 (confirmed by sequencing) was used as template to amplify the fragement 1.2.
 +
also we amplify the plasmids extracted from 2 colonies contrain the plasmid coding dCas Nm ordered from adgene.
  
 +
Q5 PCR was performed on plasmids with the following protocol:
  
−
[[File:T--Paris_Saclay--20160824_PCR_transfo_GFP_PSB1C3.jpg|400px|thumb|right|Clone 2-4-7-11 are correct.]]
+
For each 50μl of reaction, mix the following reagents :
 +
* 1 µL of plasmid
 +
* 1 µL of dNTPs (10mM)
 +
* 1 µL of each primer mix (10µM)
 +
* 10 µL of Q5 buffer (5X)
 +
* 0,5 µL of Q5 high fidelity polymerase
 +
* 35,5 µL of nuclease free water
  
 +
Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice.
 +
Perform PCR as follow:
 +
{| class="wikitable"
 +
|-
 +
!Step
 +
!Temperature
 +
!Time
 +
|-
 +
|Initial denaturation
 +
|98°C
 +
|30sec
 +
|-
 +
|rowspan="3"|30 cycles
 +
|98°C
 +
|10sec
 +
|-
 +
|Tm
 +
|20sec
 +
|-
 +
|72°C
 +
|t
 +
|-
 +
|Final Extension
 +
|72°C
 +
|2min
 +
|-
 +
|Hold
 +
|4°C
 +
|$\infinity\$
 +
|}
  
 +
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
  
 +
{| class="wikitable"
 +
|-
 +
!Matrix
 +
!gblock1.2 in pJET
 +
!plasmid adgene contain dCas9 Nm clones 1 and 2
 +
|-
 +
|Primers
 +
|iPS121 and iPS122
 +
|iPS121 and iPS122
 +
|-
 +
|Tm
 +
|72°C
 +
|72°C
 +
|-
 +
|t
 +
|30 sec
 +
|30 sec
 +
|}
  
 +
[[File:T--Paris_saclay--2408.extension.png|400px|thumb|center|Result of migration ]]
  
 +
====NanoDrop Measurements====
 +
''By Mahnaz''
  
 +
{| class="wikitable"
 +
!Sample
 +
!Concentration (ng/µL)
 +
|-
 +
|PCR fragment 1.2 <div id="CR fragment 1.2"></div>
 +
|69.08
 +
|-
 +
|}
  
 +
==== gBlock 1.1 and gblock 1.2  Ligation====
 +
''By Mahnaz''
  
 +
gBlock 1.1 and gblock 1.2 were ligated together as following :
 +
* 8µL of gBlock 1.1 PCR product from the 04/08/2016
 +
* 8µM of gblock 1.2 PCR product from 24/08/2016
 +
* 2µL of Buffer T4 10X
 +
* 2µL of ligase T4 enzyme
  
 +
The ligation product was put at 4°c overnight.
  
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 16:59, 9 October 2016

Wednesday 24th August

Visualization

Gel Electrophoresis of Gibson Assembly's products(fragment 3-4) and GFP 1-9 cloned in PSB1C3

By Terrence

Colony PCR of 12 colonies transformed with Gibson Assembly products (fragment 3-4).
Colony PCR of 12 colonies containing the GFP 1-9.
- Clones 2, 4, 7 and 11 are as expected for GFP 1.9.

pPS16_002 (1.2) clones 8 and 14 and pPS16_014(SgRna Nm) clone 12 Extraction

By Terrence

The extraction was carried out following the usual protocol.


Result of the extraction


Nano drop :

concentration (ng/µL) 260/230 260/280
1.2.14 47.81 1.64 1.84
1.2.8 834.5 2.40 1.99
SgRna Nm 12 124.21 2.00 1.89

Transformation of gblockFRB in pJET and gblock Sg_RNA_NM in pJET

By Terrence


First we made a ligation with

Component Volume (µL)
Reaction Buffer 10
PCR Product (FRB or Nm) 1
pJET1.2/blunt 1
water, nuclease free 7
T4 DNA Ligase 1

Then we transformed 2µL of ligation product into 50 µL of DH5a competent cells, following the usual protocol.

Transformation products were plated with three different volume : 50µL , 150µl and 350µL. 350µL only for the control.

Plasmid DNA extraction (Midi Prep)

By Léa & Manhaz

We have decided to use the plasmid coding dCas9 Nm to amplify the fragement corresponding g-block 1.2. 200 mL of transformed DH5a cells with Adgene dCas9 Nm plasmids (clone 1 and 2) cultures were extracted usind a Midi Prep kit. It was resuspended into 50µL of sterile water.

The plasmid then reserved in -20.

PCR Q5 on plasmid

By Léa & Manhaz

Plasmid pJet cotnains gblock 1.2 (confirmed by sequencing) was used as template to amplify the fragement 1.2. also we amplify the plasmids extracted from 2 colonies contrain the plasmid coding dCas Nm ordered from adgene.

Q5 PCR was performed on plasmids with the following protocol:

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

  • 1 µL of plasmid
  • 1 µL of dNTPs (10mM)
  • 1 µL of each primer mix (10µM)
  • 10 µL of Q5 buffer (5X)
  • 0,5 µL of Q5 high fidelity polymerase
  • 35,5 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 98°C 30sec
30 cycles 98°C 10sec
Tm 20sec
72°C t
Final Extension 72°C 2min
Hold 4°C $\infinity\$

Primers used were:

Matrix gblock1.2 in pJET plasmid adgene contain dCas9 Nm clones 1 and 2
Primers iPS121 and iPS122 iPS121 and iPS122
Tm 72°C 72°C
t 30 sec 30 sec
Result of migration

NanoDrop Measurements

By Mahnaz

Sample Concentration (ng/µL)
PCR fragment 1.2
69.08

gBlock 1.1 and gblock 1.2 Ligation

By Mahnaz

gBlock 1.1 and gblock 1.2 were ligated together as following :

  • 8µL of gBlock 1.1 PCR product from the 04/08/2016
  • 8µM of gblock 1.2 PCR product from 24/08/2016
  • 2µL of Buffer T4 10X
  • 2µL of ligase T4 enzyme

The ligation product was put at 4°c overnight.






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