Difference between revisions of "Team:Paris Saclay/Notebook/August/11"

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= Thursday 11<sup>th</sup> August=
 
= Thursday 11<sup>th</sup> August=
−
==Lab work==
+
 
 
===Visualization===
 
===Visualization===
−
====Transformation of DH5a cells with pPS16_009====
+
====Transformation of DH5a cells with pPS16_020====
 
''By Léa''
 
''By Léa''
  
−
Dh5a cells were transformed with pPS16_009, or psB1c3 (control), or not transformed (control)using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]].
+
Dh5a cells were transformed with pPS16_020 (GFP 1-9 ligated with pSB1C3 digested), or the linearized pSB1C3 (control), or not transformed (control) using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]].
−
 
+
  
 
====[[Team:Paris_Saclay/Experiments#pPS16_010|pPS16_010]] extraction====
 
====[[Team:Paris_Saclay/Experiments#pPS16_010|pPS16_010]] extraction====
 
''By Alice''
 
''By Alice''
  
−
After screening colony screening PCR, 3 clones (clones 3, 7 and 8) transformed with pPS16_010 seemed to have the insert of interest. Plasmids from these clones were extracted following [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|this protocol]]. After extraction, plasmids were migrated on a gel.z
+
After colony screening PCR, 3 clones (clones 3, 7 and 8) seemed to have the insert of interest. Plasmids from these clones were extracted following [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|this protocol]]. After extraction, plasmids were migrated on a gel.  
 +
 
 +
Band size expected:
 +
{| class="wikitable"
 +
|-
 +
!Plasmid name
 +
!Plasmid size (bp)
 +
|-
 +
|pPS16_010
 +
|3070
 +
|}
 +
 
 +
 
[[File:T--Paris_Saclay--20160811_extraction_FRB_3_7_8.jpeg|400px|thumb|right|Migration of FRB plasmid]]
 
[[File:T--Paris_Saclay--20160811_extraction_FRB_3_7_8.jpeg|400px|thumb|right|Migration of FRB plasmid]]
  
−
====DreamTaq PCR of transformed cell with pPS16_002,008,010, 011, 012, 013, 014 and puC 19====
+
 
 +
 
 +
Nano Drop:
 +
 
 +
{| class="wikitable"
 +
|-
 +
!Plasmid name
 +
!Concentration (ng/µL)
 +
!260/230
 +
!260/280
 +
|-
 +
|pPS16_010 clone 3
 +
|334.48
 +
|2.35
 +
|1.94
 +
|-
 +
|pPS16_010 clone 7
 +
|321.06
 +
|2.32
 +
|1.98
 +
|-
 +
|pPS16_010 clone 8
 +
|139.13
 +
|2.10
 +
|1.88
 +
|}
 +
 
 +
 
 +
==== Extraction of pPS16_002 (clone 7), pPS16_011 (clones 3, 4, 6), pPS16_014 (clones 11) and pPS16_009(clone 7)  ====
 +
''By Terrence''
 +
 
 +
The extraction was carried out following the usual  [[Team:Paris_Saclay/Experiments#DNA_extraction_using_the_Invitrogen_ChargeSwitch.C2.AE-Pro_Plasmid_Miniprep_Kit|protocol]].
 +
 
 +
[[File:T--Paris_Saclay--extraction_20160811.jpg|400px|thumb|right|Extraction of pPS16_002 (clone 7), pPS16_011 (clones 3, 4, 6), pPS16_014 (clones 3 and 11) and pPS16_009 (clone 7) ]]
 +
 
 +
 
 +
 
 +
Nano Drop:
 +
 
 +
{| class="wikitable"
 +
|-
 +
!Plasmid name
 +
!Concentration (ng/µL)
 +
!260/230
 +
!260/280
 +
|-
 +
|pPS16_011 clone 3
 +
|34.08
 +
|1.54
 +
|1.57
 +
|-
 +
|pPS16_011 clone 4
 +
|368.42
 +
|2.36
 +
|1.92
 +
|-
 +
|pPS16_011 clone 6
 +
|295.99
 +
|2.29
 +
|1.91
 +
|-
 +
|pPS16_014 clone 11
 +
|529.17
 +
|2.39
 +
|1.91
 +
|-
 +
|pPS16_002 clone 7
 +
|30.50
 +
|1.76
 +
|2.13
 +
|-
 +
|pPS16_009 clone 7
 +
|282.1
 +
|2.31
 +
|1.93
 +
 
 +
|}
 +
 
 +
====High Fidelity Phusion PCR of transformed cell with ligation 2 and 3====
 +
''By Laetitia''
 +
 
 +
Phusion PCR was performed on the ligation product 2 (gBlock 2.1 ligated with gBblock 2.2) and the ligation product 3 (gBlock 3.1 ligated with gBlock 3.2) made on 09/08/16.
 +
It was done following [[Team:Paris_Saclay/Experiments#phusionPCR|this protocol]].
 +
For the PCR on ligation 2 the primers used were IPS 123 and IPS 84.
 +
For the PCR on ligation 3 the primers used were IPS 128 and IPS 83.
 +
 
 +
Gel1
 +
 
 +
====DreamTaq PCR of transformed cell with pPS16_002,008,010, 011, 012, 013, 014 and puC19====
 
''By Léa and Laetitia''
 
''By Léa and Laetitia''
  
−
PCR DreamTaq was performed on 6 clones of each transformed celles from 10/08/16.
+
PCR DreamTaq was performed on 6 clones of each transformed cells from 10/08/16.
−
Each clone was plated and put in liquid culture with LB and Ampicillin.
+
Each clone was spread and put in liquid culture with LB and Ampicillin.
 +
No PCR products were observed on the gel.
  
 
====Phusion PCR on PSB1C3====
 
====Phusion PCR on PSB1C3====
 
''By Alice''
 
''By Alice''
  
−
PSB1C3 was amplified with Phusion DNA polymerase following [[Team:Paris_Saclay/Experiments#phusionPCR|this protocol]]. Two primers ([[Team:Paris_Saclay/Experiments#primers|iPS41 and iPS42]]) were chosen. Annealing temperature was 70.9°C. Elongation step lasted 1min. After amplification, 5 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.
+
pSB1C3 was amplified with Phusion polymerase following [[Team:Paris_Saclay/Experiments#phusionPCR|this protocol]]. Two primers ([[Team:Paris_Saclay/Experiments#primers|iPS41 and iPS42]]) were used. Annealing temperature was 70.9°C. After amplification, 5 µL of each PCR products with loading dye and 10 µL of DNA ladder were placed on gel and migrated at 100v during 30 min.
  
 
PCR products expected:
 
PCR products expected:
Line 37: Line 137:
 
|2070
 
|2070
 
|}
 
|}
 +
[[File:T--Paris_Saclay--20160812_PCR_PSB1C3.jpeg|500px|thumb|right|Migration of PSB1C3 amplification]]
  
  
 +
====Result of the ligation 2-3 PCR.====
 +
''By Caroline''
 +
 +
We made the PCR again as the previous result was not satisfying.
 +
Ligation product were cultivate overnight.
 +
[[File:T--Paris_Saclay--extraction_20160811_pcr_ligation2-3.jpg|500px|thumb|right|Result of the ligation 2-3 PCR]]
 +
 +
===Bringing DNA closer ===
 +
==== Extraction of DS-SPcasN- and DS-TDcasN- ====
 +
''By Terrence''
 +
 +
The extraction was carried out with the Nucleobond Ax Kit.
 +
 +
[[File:T--Paris_Saclay--extraction_20160811_SP_TD_echelle.jpeg|400px|thumb|right|Extraction of DS-SPcasN-  and DS-TDcasN-]]
 +
 +
Nano Drop:
 +
 +
{| class="wikitable"
 +
|-
 +
!Plasmid name
 +
!Concentration (ng/µL)
 +
!260/230
 +
!260/280
 +
|-
 +
|DS-SPcasN-
 +
|185.46
 +
|2.23
 +
|1.79
 +
|-
 +
|DS-TDcasN
 +
|198.97
 +
|1.88
 +
|1.79
 +
 +
|}
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 16:37, 9 October 2016

Thursday 11th August

Visualization

Transformation of DH5a cells with pPS16_020

By Léa

Dh5a cells were transformed with pPS16_020 (GFP 1-9 ligated with pSB1C3 digested), or the linearized pSB1C3 (control), or not transformed (control) using the usual protocol.

pPS16_010 extraction

By Alice

After colony screening PCR, 3 clones (clones 3, 7 and 8) seemed to have the insert of interest. Plasmids from these clones were extracted following this protocol. After extraction, plasmids were migrated on a gel.

Band size expected:

Plasmid name Plasmid size (bp)
pPS16_010 3070


Migration of FRB plasmid


Nano Drop:

Plasmid name Concentration (ng/µL) 260/230 260/280
pPS16_010 clone 3 334.48 2.35 1.94
pPS16_010 clone 7 321.06 2.32 1.98
pPS16_010 clone 8 139.13 2.10 1.88


Extraction of pPS16_002 (clone 7), pPS16_011 (clones 3, 4, 6), pPS16_014 (clones 11) and pPS16_009(clone 7)

By Terrence

The extraction was carried out following the usual protocol.

Extraction of pPS16_002 (clone 7), pPS16_011 (clones 3, 4, 6), pPS16_014 (clones 3 and 11) and pPS16_009 (clone 7)


Nano Drop:

Plasmid name Concentration (ng/µL) 260/230 260/280
pPS16_011 clone 3 34.08 1.54 1.57
pPS16_011 clone 4 368.42 2.36 1.92
pPS16_011 clone 6 295.99 2.29 1.91
pPS16_014 clone 11 529.17 2.39 1.91
pPS16_002 clone 7 30.50 1.76 2.13
pPS16_009 clone 7 282.1 2.31 1.93

High Fidelity Phusion PCR of transformed cell with ligation 2 and 3

By Laetitia

Phusion PCR was performed on the ligation product 2 (gBlock 2.1 ligated with gBblock 2.2) and the ligation product 3 (gBlock 3.1 ligated with gBlock 3.2) made on 09/08/16. It was done following this protocol. For the PCR on ligation 2 the primers used were IPS 123 and IPS 84. For the PCR on ligation 3 the primers used were IPS 128 and IPS 83.

Gel1

DreamTaq PCR of transformed cell with pPS16_002,008,010, 011, 012, 013, 014 and puC19

By Léa and Laetitia

PCR DreamTaq was performed on 6 clones of each transformed cells from 10/08/16. Each clone was spread and put in liquid culture with LB and Ampicillin. No PCR products were observed on the gel.

Phusion PCR on PSB1C3

By Alice

pSB1C3 was amplified with Phusion polymerase following this protocol. Two primers (iPS41 and iPS42) were used. Annealing temperature was 70.9°C. After amplification, 5 µL of each PCR products with loading dye and 10 µL of DNA ladder were placed on gel and migrated at 100v during 30 min.

PCR products expected:

Plasmids expected band size (bp)
PSB1C3 2070
Migration of PSB1C3 amplification


Result of the ligation 2-3 PCR.

By Caroline

We made the PCR again as the previous result was not satisfying. Ligation product were cultivate overnight.

Result of the ligation 2-3 PCR

Bringing DNA closer

Extraction of DS-SPcasN- and DS-TDcasN-

By Terrence

The extraction was carried out with the Nucleobond Ax Kit.

Extraction of DS-SPcasN- and DS-TDcasN-

Nano Drop:

Plasmid name Concentration (ng/µL) 260/230 260/280
DS-SPcasN- 185.46 2.23 1.79
DS-TDcasN 198.97 1.88 1.79





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