Difference between revisions of "Team:Paris Saclay/Notebook/September/7"

(→‎Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3)
(→‎Lab work)
 
(19 intermediate revisions by 3 users not shown)
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{{Team:Paris_Saclay/notebook_header}}
 +
 
= Wednesday 7<sup>th</sup> September=
 
= Wednesday 7<sup>th</sup> September=
−
==Lab work==
+
 
 
===Visualization===
 
===Visualization===
  
 
====Gibson of cleaned up PCR products FRB from clone 9 x pSB1C3 - GFP 11 from clone 8 ====
 
====Gibson of cleaned up PCR products FRB from clone 9 x pSB1C3 - GFP 11 from clone 8 ====
−
''By Maxence''
+
''By Maxence & Mahnaz''
  
−
Gibson was performed on cleaned up PCR products FRB from clone 9 (insert) x pSB1C3 - GFP 11 from clone 8 (plasmid) with the following protocol:
+
Gibson was performed with cleaned up PCR products FRB from clone 9 (insert) x pSB1C3 - GFP 11 from clone 8 (plasmid) (obtained the 6th September) with the following protocol:
  
 
For each 20μl of reaction, mix the following reagents :
 
For each 20μl of reaction, mix the following reagents :
Line 12: Line 14:
 
* 3.68 µL of plasmid
 
* 3.68 µL of plasmid
 
* 5.67 µL of water
 
* 5.67 µL of water
−
* 10 µL of buffer
+
* 10 µL of buffer mix
  
−
Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL. The PCR was performed as follow : 1 hour at 50°C.
+
Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). The PCR was performed as follow : 1 hour at 50°C.
  
−
====Clean-up of Gibson products ====
+
====Transformation of DH5a cells with FRB - GFP 11 in pSB1C3 (pPS16_019) obtained by Gibson====
−
''By Maxence''
+
''By Maxence & Mahnaz''
  
−
Gibson products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]].
+
Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11 (pPS16_019), or controls (no buffer mix and plasmid alone) using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]].
  
−
====Transformation of DH5a cells with FRB - GFP 11 in pSB1C3 obtained by Gibson====
+
====Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3 (pPS16_016)====
−
''By Maxence''
+
''By Maxence & Mahnaz''
−
 
+
−
Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11, or containing control (no mix added when Gibson performed) using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]].
+
−
 
+
−
====Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3====
+
−
''Maxence''
+
  
 
Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 16 clones containing dCas9 NM - GFP 10 obtained by Q5 amplification and 16 clones containing dCas9 NM - GFP 10 obtained by Phusion amplification  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 + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.
 
Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 16 clones containing dCas9 NM - GFP 10 obtained by Q5 amplification and 16 clones containing dCas9 NM - GFP 10 obtained by Phusion amplification  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 + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.
Line 52: Line 49:
 
|30 sec
 
|30 sec
 
|-
 
|-
−
|Tm
+
|63.6°C
 
|30 sec
 
|30 sec
 
|-
 
|-
 
|72°C
 
|72°C
−
|t
+
|1 min 30 sec
 
|-
 
|-
 
|Final Extension
 
|Final Extension
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|}
 
|}
  
−
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
+
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
  
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
 
!Matrix
 
!Matrix
−
!Clones containing dCas9 NM - GFP 10 in pSB1C3
+
!Clones containing dCas9 NM - GFP 10 in pSB1C3 (pPS16_016)
 
|-
 
|-
 
|Primers
 
|Primers
 
|iPS83 and iPS84
 
|iPS83 and iPS84
−
|-
 
−
|Tm
 
−
|63.6°C
 
−
|-
 
−
|t
 
−
|1 min 30
 
 
|}
 
|}
  
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|}
 
|}
  
−
[[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]]
+
[[File:T--Paris Saclay--Gel112.png|400px|thumb|center|Result of the migration - Phusion]]
 +
[[File:T--Paris Saclay--Gel113.png|400px|thumb|center|Result of the migration - Q5 Pol]]
  
−
GEL GEL GEL
+
Only PCR products around 650 pb were obtained for both Pol. This kind of results has already been obtained by the past and the experience has to be improved.
  
−
====Q5 PCR on dCas9 ST - GFP 11 in pSB1C3, FRB in pJET and GFP 1.9 in pUC19 ====
+
====Samples preparation for sequencing====
−
''By Maxence''
+
''By Maxence & Mahnaz''
−
 
+
−
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:
+
−
{| class="wikitable"
+
−
|-
+
−
!Step
+
−
!Temperature
+
−
!Time
+
−
|-
+
−
|Initial denaturation
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−
|98°C
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−
|30sec
+
−
|-
+
−
|rowspan="3"|30 cycles
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−
|98°C
+
−
|10sec
+
−
|-
+
−
|Tm
+
−
|20sec
+
−
|-
+
−
|72°C
+
−
|t
+
−
|-
+
−
|Final Extension
+
−
|72°C
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−
|2min
+
−
|-
+
−
|Hold
+
−
|4°C
+
−
|$\infinity\$
+
−
|}
+
−
 
+
−
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
+
−
 
+
−
{| class="wikitable"
+
−
|-
+
−
!Matrix
+
−
!dCas9 ST - GFP 11 in pSB1C3 clones 6 and 8
+
−
!FRB in pJET clones 4 and 9
+
−
!GFP 1.9 in pUC19
+
−
|-
+
−
|Primers
+
−
|iPS152 and iPS151
+
−
|iPS149 and iPS150
+
−
|iPS84 and iPS140
+
−
|-
+
−
|Tm
+
−
|57,5°C
+
−
|56,7°C
+
−
|72°C
+
−
|-
+
−
|t
+
−
|1 min 30
+
−
|20 sec
+
−
|30 sec
+
−
|}
+
−
 
+
−
 
+
−
====NanoDrop Measurements====
+
−
''By Maxence''
+
−
 
+
−
{| class="wikitable"
+
−
!Sample
+
−
!Concentration (ng/µL)
+
−
|-
+
−
|PCR fragment GFP 11 clone 6<div id="PCR fragment GFP 11 - pSB1C3 clones 6"></div>
+
−
|187.23
+
−
|-
+
−
|PCR fragment GFP 11 clone 8<div id="PCR fragment GFP 11 - pSB1C3 clones 8"></div>
+
−
|156.85
+
−
|-
+
−
|PCR fragment FRB clone 4<div id="PCR fragment FRB clone 4"></div>
+
−
|75.67
+
−
|-
+
−
|PCR fragment FRB clone 9<div id="PCR fragment FRB clone 9"></div>
+
−
|246.41
+
−
|-
+
−
|PCR fragment GFP 1.9<div id="PCR fragment GFP 1.9"></div>
+
−
|22.06
+
−
|-
+
−
|}
+
−
 
+
−
====Gel of cleaned up PCR products====
+
−
''By Maxence''
+
−
 
+
−
After amplification, 3 µL of each cleaned up 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)
+
−
|-
+
−
|GFP 11- pSB1C3
+
−
|2500
+
−
|-
+
−
|FRB
+
−
|374
+
−
|-
+
−
|GFP 1.9
+
−
|862
+
−
|}
+
  
−
[[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]]
+
20 µL of plasmides dCas9 ST - GFP 11 (pPS16_016) (clones 6 and 8) were sent to be sequenced. 20 µL of the primers iPS168 (5µM), iPS169 (5µM) and iPS171 (5µM) were sent for sequencing.
  
−
GEL GEL GEL
+
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 17:03, 9 October 2016

Wednesday 7th September

Visualization

Gibson of cleaned up PCR products FRB from clone 9 x pSB1C3 - GFP 11 from clone 8

By Maxence & Mahnaz

Gibson was performed with cleaned up PCR products FRB from clone 9 (insert) x pSB1C3 - GFP 11 from clone 8 (plasmid) (obtained the 6th September) with the following protocol:

For each 20μl of reaction, mix the following reagents :

  • 0.64 µL of insert
  • 3.68 µL of plasmid
  • 5.67 µL of water
  • 10 µL of buffer mix

Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). The PCR was performed as follow : 1 hour at 50°C.

Transformation of DH5a cells with FRB - GFP 11 in pSB1C3 (pPS16_019) obtained by Gibson

By Maxence & Mahnaz

Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11 (pPS16_019), or controls (no buffer mix and plasmid alone) using the usual protocol.

Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3 (pPS16_016)

By Maxence & Mahnaz

Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 16 clones containing dCas9 NM - GFP 10 obtained by Q5 amplification and 16 clones containing dCas9 NM - GFP 10 obtained by Phusion amplification were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) 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)
  • 1 µL of each primer mix (10µM)
  • 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step Temperature Time
Initial denaturation 95°C 3 min
30 cycles 95°C 30 sec
63.6°C 30 sec
72°C 1 min 30 sec
Final Extension 72°C 7 min
Hold 4°C $\infinity\$

Primers used were:

Matrix Clones containing dCas9 NM - GFP 10 in pSB1C3 (pPS16_016)
Primers iPS83 and iPS84

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)
dCas9 NM - GFP 10 - pSB1C3 3688
Result of the migration - Phusion
Result of the migration - Q5 Pol

Only PCR products around 650 pb were obtained for both Pol. This kind of results has already been obtained by the past and the experience has to be improved.

Samples preparation for sequencing

By Maxence & Mahnaz

20 µL of plasmides dCas9 ST - GFP 11 (pPS16_016) (clones 6 and 8) were sent to be sequenced. 20 µL of the primers iPS168 (5µM), iPS169 (5µM) and iPS171 (5µM) were sent for sequencing.





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