Difference between revisions of "Team:Paris Saclay/Notebook/August/22"

(→‎Purification of PSB1C3 with kit)
(→‎Lab work)
 
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{{Team:Paris_Saclay/notebook_header}}
 
{{Team:Paris_Saclay/notebook_header}}
 
= Monday 22<sup>th</sup> August=
 
= Monday 22<sup>th</sup> August=
−
==Lab work==
+
 
 
===Visualization===
 
===Visualization===
−
====Low fidelity Dreamn Taq PCR of pPS16_016====
+
====Dream Taq PCR of GFP1-9 cloned in the pSB1C3====
 
''By Mathilde''
 
''By Mathilde''
  
−
Clones 7,8,9 and 10 of pPS16_016 were amplified following the protocol :
+
Clones 7,8,9 and 10 of GFP1-9 cloned in the pSB1C3 were amplified following the protocol :
−
* 1,5µL of Green Buffer
+
* 1,5µL of DreamTaq Buffer
 
* 1µL of dNTPs
 
* 1µL of dNTPs
−
* 1µL of each primers IPS83 and IPS84
+
* 1µL of each primers IPS83 and IPS84 (10 µM)
 
* 0,13µL of Dream Taq Polymerase
 
* 0,13µL of Dream Taq Polymerase
 
* 19,37 µL of H20  
 
* 19,37 µL of H20  
  
−
Colonies were put into sterile water, put through 5 minutes of denaturation before the addition of the mix.
+
Colonies were put into sterile water, with 5 minutes of denaturation (95°C in the PCR machine) before the addition of the mix.
 
Annealing temperature was 57°c.
 
Annealing temperature was 57°c.
−
After amplification, 1µL of each PCR product and 10µL of the DNA purple ladder were palced in wells and migrated at 100V for 30min.
+
After amplification, 1µL of each PCR product and 10µL of the DNA purple ladder were loaded into a 0,8% agarose gel + BET and then migrated at 100V for 30min.
 +
 
 +
 
 +
[[File:T--Paris_Saclay--160822_gel1.JPG|400px|thumb|right|Result of the PCR]]
  
−
====Sequencing of 1.2, FKBP, FRB====
+
====Sequencing of 1.2, FKBP, FRB cloned in pJET plasmid====
 
''Terrence and Léa''
 
''Terrence and Léa''
  
Line 64: Line 67:
 
''Terrence and Mathilde''
 
''Terrence and Mathilde''
  
−
After amplification, 50µL of each PCR product diluted 6 tumes with 10 µL of the gel loadding dye, and 10µL of the DNA purple ladder were placed in wells and migrated at 100V for 30min.
+
After amplification, 50µL of each PCR product diluted 6 times with 10 µL of the gel loading dye, and 10µL of the DNA purple ladder were put into a 0,8 agarose gel + BET and migrated at 100V for 30min.
 
The extraction was carried out with the [[Team:Paris_Saclay/Experiments#DNA_extraction_from_agarose_gels_with_NucleoSpin_Gel_and_PCR_Clean-up|usual protocol]].
 
The extraction was carried out with the [[Team:Paris_Saclay/Experiments#DNA_extraction_from_agarose_gels_with_NucleoSpin_Gel_and_PCR_Clean-up|usual protocol]].
  
Line 89: Line 92:
 
|}
 
|}
  
 +
[[File:T--Paris_Saclay--160822_gel_extraction.JPG|400px|thumb|right|Gel for the extraction]]
  
 
====Purification of PSB1C3 with kit====
 
====Purification of PSB1C3 with kit====
 
''Terrence''
 
''Terrence''
  
−
PSB1C3 was purified following the [[Team:Paris_Saclay/Experiments##Purification_:_PCR_clean-up_with_NucleoSpin_Gel_and_PCR_Clean-up|usual protocol]].
+
pSB1C3 was purified following the [[Team:Paris_Saclay/Experiments##Purification_:_PCR_clean-up_with_NucleoSpin_Gel_and_PCR_Clean-up|usual protocol]].
−
To be sure that PSB1C3 were linear, it was digested with DPN1.
+
To remove any circular plasmid pSB1C3, it was digested with the enzyme DPNI.
  
 
Nano Drop :  
 
Nano Drop :  
Line 100: Line 104:
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
−
!  
+
!
 
!size (ng/µL)
 
!size (ng/µL)
 
!260/230
 
!260/230
Line 113: Line 117:
  
  
 +
====Gibson on fragment 3 and fragment 4====
 +
''Terrence''
 +
 +
We have assembled the fragment 3 and 4 following the <html>  <a href="https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol">HiFi assembly protocol</a></html>with 50ng of vector and 2 times more of insert.
 +
Here is the volume used for the Gibson :
 +
{| class="wikitable"
 +
|-
 +
!Components
 +
!Volume (µL)
 +
|-
 +
|PSB1C2
 +
|0.85
 +
|-
 +
|Fragment 3
 +
|6.83
 +
|-
 +
|Fragment 4
 +
|1.60
 +
|-
 +
|H2O
 +
|0.72
 +
|-
 +
|}
 +
We made 2 tubes composed of this upper mix.
 +
In the control one, we added 10 µL of water, whereas in the "Gibson" one, we added 10µL of Gibson mix.
 +
 +
 +
Gibson assembly product (fragments 3 and 4 ligation) and control were transformed into DH5a cells, using 2 µL of DNA, and following the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]].
 +
 +
====GFP1-9 and psB1C3 ligation product transformation in DH5a====
 +
''By Léa''
 +
 +
The GFP1-9 and pSB1C3 ligation products of the 10/08/2016 was transformed into DH5a cells, using 2 µL of DNA, and following the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]].
 +
Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C o/n.
 +
 +
===Interlab Study===
 +
 +
====Transformation into DH5a cells====
 +
''By Léa''
 +
 +
Devices 2 and 3, negative control and positive control were transformed into DH5a cells, following the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]].
 +
Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C o/n.
 +
 +
====Device 1 culture====
 +
''By Léa''
 +
 +
Device 1 transformed DH5a glycerol stock was plated on LB Agar chloramphenicol Petri Dishes and incubated at 37°C o/n.
 +
 +
====Cell culture====
 +
''By Mahnaz''
 +
 +
Bacteria containing plasmids coding NM Cas9 ([https://www.addgene.org/48646/ DS-NMcas])which have been ordered from Addgene were put into 3mL of LB medium containing 50µg/mL of spectinomycin and incubated overnight at 37°C, 180 rpm.
  
  
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 16:58, 9 October 2016

Monday 22th August

Visualization

Dream Taq PCR of GFP1-9 cloned in the pSB1C3

By Mathilde

Clones 7,8,9 and 10 of GFP1-9 cloned in the pSB1C3 were amplified following the protocol :

  • 1,5µL of DreamTaq Buffer
  • 1µL of dNTPs
  • 1µL of each primers IPS83 and IPS84 (10 µM)
  • 0,13µL of Dream Taq Polymerase
  • 19,37 µL of H20

Colonies were put into sterile water, with 5 minutes of denaturation (95°C in the PCR machine) before the addition of the mix. Annealing temperature was 57°c. After amplification, 1µL of each PCR product and 10µL of the DNA purple ladder were loaded into a 0,8% agarose gel + BET and then migrated at 100V for 30min.


Result of the PCR

Sequencing of 1.2, FKBP, FRB cloned in pJET plasmid

Terrence and Léa

Was sent :

Clone Concentration size
1.2 11 392.66 3934
FKBP 3 272.37 3393
FKBP 4 404.65 3393
FKBP 5 204.87 3393
FRB 2 256.44 3347
FRB 5 129.86 3347

Purification on gel of gBlocks 3 and 4

Terrence and Mathilde

After amplification, 50µL of each PCR product diluted 6 times with 10 µL of the gel loading dye, and 10µL of the DNA purple ladder were put into a 0,8 agarose gel + BET and migrated at 100V for 30min. The extraction was carried out with the usual protocol.

Nano Drop :


size (ng/µL) 260/230 260/280
Fragment 3 11.07 0.48 3.02
Fragment 4 50.12 1.07 1.93
Gel for the extraction

Purification of PSB1C3 with kit

Terrence

pSB1C3 was purified following the usual protocol. To remove any circular plasmid pSB1C3, it was digested with the enzyme DPNI.

Nano Drop :

size (ng/µL) 260/230 260/280
PSB1C3 58.66 0.74 1.71


Gibson on fragment 3 and fragment 4

Terrence

We have assembled the fragment 3 and 4 following the HiFi assembly protocolwith 50ng of vector and 2 times more of insert. Here is the volume used for the Gibson :

Components Volume (µL)
PSB1C2 0.85
Fragment 3 6.83
Fragment 4 1.60
H2O 0.72

We made 2 tubes composed of this upper mix. In the control one, we added 10 µL of water, whereas in the "Gibson" one, we added 10µL of Gibson mix.


Gibson assembly product (fragments 3 and 4 ligation) and control were transformed into DH5a cells, using 2 µL of DNA, and following the usual protocol.

GFP1-9 and psB1C3 ligation product transformation in DH5a

By Léa

The GFP1-9 and pSB1C3 ligation products of the 10/08/2016 was transformed into DH5a cells, using 2 µL of DNA, and following the usual protocol. Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C o/n.

Interlab Study

Transformation into DH5a cells

By Léa

Devices 2 and 3, negative control and positive control were transformed into DH5a cells, following the usual protocol. Transformation products were plated on LB Agar chloramphenicol Petri dishes and incubated at 37°C o/n.

Device 1 culture

By Léa

Device 1 transformed DH5a glycerol stock was plated on LB Agar chloramphenicol Petri Dishes and incubated at 37°C o/n.

Cell culture

By Mahnaz

Bacteria containing plasmids coding NM Cas9 (DS-NMcas)which have been ordered from Addgene were put into 3mL of LB medium containing 50µg/mL of spectinomycin and incubated overnight at 37°C, 180 rpm.






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