Difference between revisions of "Team:Paris Saclay/Notebook/September/8"

(→‎Gibson of cleaned up PCR products FRB from clone 9 x pSB1C3 - GFP 11 from clone 8)
(→‎Lab work)
 
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 +
{{Team:Paris_Saclay/notebook_header}}
 +
 
= Thursday 8<sup>th</sup> September=
 
= Thursday 8<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''
  
 
As no colonies were obtained after one night culture, a new 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.
 
As no colonies were obtained after one night culture, a new 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.
Line 16: Line 18:
 
Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). Two different buffer mix were tested (the usual one and another one from Philippe). 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). Two different buffer mix were tested (the usual one and another one from Philippe). The PCR was performed as follow : 1 hour at 50°C.
  
−
====Transformation of DH5a cells with FRB - GFP 11 in pSB1C3 obtained by Gibson====
+
====Transformation of DH5a cells with FRB - GFP 11 in pSB1C3 (pPS16_019) obtained by Gibson====
−
''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]].
+
Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11 (pPS16_019), or containing controls (no buffer mix and plasmid alone) using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]]. As mentioned before, two buffer mix were used. Furthermore, two different DH5a strains were tested (the usual one and another one from Philippe) and at least 12 transformants were cultured overnight : 3 (1 Gibson and 2 controls) x 2 buffer mix x 2 DH5a strains.
  
−
====Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3====
+
====Q5 PCR on FKBP in pJET and gblock 2.2 (part of dCas9 NM - GFP 10) for new clonage strategy ====
−
''Maxence''
+
''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 [[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.
+
An other clonage strategy was performed by using Gibson between FKBP, gblock 2.2 (part of dCas9 NM - GFP 10) and pSB1C3 (the one from dCas9 ST - GFP 11) in order to have FKBP - GFP 10 in pSB1C3 (pPS16_018). For that purpose, Q5 PCR was performed on plasmids with the following protocol.
  
−
For each clones contained in 20 μl water, 4.13 μL of the following mix were added :
+
For each 50μl of reaction, mix the following reagents :
−
* 2.5 µL DreamTaq Buffer
+
* 1 µL of plasmid
−
* 0.5 µL of dNTPs (10mM)
+
* 1 µL of dNTPs (10mM)
−
* 1 µL of each primer mix (10µM)
+
* 2.5 µL of each primer mix (10µM)
−
* 0.13 μl of DreamTaq Pol
+
* 10 µL of Q5 buffer (5X)
 +
* 0,5 µL of Q5 high fidelity polymerase
 +
* 32,5 µL of nuclease free water
  
−
PCR was performed as follow:  
+
Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice.
 +
Perform PCR as follow:  
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
Line 40: Line 45:
 
|-
 
|-
 
|Initial denaturation
 
|Initial denaturation
−
|95°C
+
|98°C
−
|3 min
+
|30sec
 
|-
 
|-
 
|rowspan="3"|30 cycles
 
|rowspan="3"|30 cycles
−
|95°C
+
|98°C
−
|30 sec
+
|10sec
 
|-
 
|-
 
|Tm
 
|Tm
−
|30 sec
+
|20sec
 
|-
 
|-
 
|72°C
 
|72°C
Line 55: Line 60:
 
|Final Extension
 
|Final Extension
 
|72°C
 
|72°C
−
|7 min
+
|2min
 
|-
 
|-
 
|Hold
 
|Hold
 
|4°C
 
|4°C
−
|$\infinity\$
+
|$\infty$
 
|}
 
|}
  
−
[[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
+
!gblock FKBP in pJET clones 3, 4 and 5
 +
!gblock 2.2 in pUC19
 
|-
 
|-
 
|Primers
 
|Primers
−
|iPS83 and iPS84
+
|iPS145 and iPS146
 +
|iPS147 and iPS84
 
|-
 
|-
 
|Tm
 
|Tm
−
|63.6°C
+
|62°C
 +
|70°C
 
|-
 
|-
 
|t
 
|t
−
|1 min 30
+
|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 on GFP 1.9 in pUC19 with 3% DMSO ====
 +
''By Maxence & Mahnaz''
 +
 
 +
As the annealing temperature (Tm) seems too high to obtain good results for GFP 1.9 in pUC19 amplification, DMSO was used in order to reduce the annealing temperature during the PCR. For that purpose, 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)
 +
* 2.5 µL of each primer mix (10µM)
 +
* 10 µL of buffer (5X)
 +
* 0,5 µL of Phusion polymerase
 +
* 31 µL of nuclease free water
 +
* 1.5 µL of DMSO
 +
 
 +
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
 +
|-
 +
|60°C
 +
|30sec
 +
|-
 +
|72°C
 +
|30sec
 +
|-
 +
|Final Extension
 +
|72°C
 +
|2min
 +
|-
 +
|Hold
 +
|4°C
 +
|$\infty$
 +
|}
 +
 
 +
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
 +
 
 +
{| class="wikitable"
 +
|-
 +
!Matrix
 +
!GFP 1.9 in pUC19
 +
|-
 +
|Primers
 +
|iPS140 and iPS84
 +
|-
 +
|}
 +
 
 +
====PCR Clean-up of PCR products ====
 +
''By Maxence & Mahnaz''
 +
 
 +
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]].
 +
 
 +
====NanoDrop Measurements====
 +
''By Maxence & Mahnaz''
 +
 
 +
{| class="wikitable"
 +
!Sample
 +
!Concentration (ng/µL)
 +
|-
 +
|PCR fragment FKBP clone 3<div id="FKBP clone 3"></div>
 +
|222.57
 +
|-
 +
|PCR fragment FKBP clone 4<div id="FKBP clone 4"></div>
 +
|219.99
 +
|-
 +
|PCR fragment FKBP clone 5<div id="FKBP clone 5"></div>
 +
|376.2
 +
|-
 +
|PCR fragment gblock 2.2<div id="gblock 2.2"></div>
 +
|276.33
 +
|-
 +
|}
 +
 
 +
====Gel of cleaned up PCR products====
 +
''By Maxence & Mahnaz''
 +
 
 +
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 :
 
PCR products expected were :
Line 88: Line 183:
 
!Expected band size (bp)
 
!Expected band size (bp)
 
|-
 
|-
−
|dCas9 NM - GFP 10 - pSB1C3
+
|FKBP
−
|3688
+
|419
 +
|-
 +
|gblock 2.2
 +
|339
 +
|-
 +
|GFP 1.9
 +
|862
 
|}
 
|}
  
−
[[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]]
+
[[File:T--Paris Saclay--Gel114.png|400px|thumb|center|Result of the migration]]
 +
 
 +
All PCR products were at the good size.
 +
 
 +
[[File:T--Paris Saclay--Gel115.png|400px|thumb|center|Result of the migration]]
 +
 
 +
All PCR product were at the good size.
  
−
GEL GEL GEL
+
[[File:T--Paris Saclay--Gel116.png|400px|thumb|center|Result of the migration]]
  
−
====Samples preparation for sequencing====
+
PCR product was at the good size.
−
''by Maxence''
+
  
−
20 µL of plasmides dCas9 ST - GFP 11 (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.
+
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 17:03, 9 October 2016

Thursday 8th September

Visualization

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

By Maxence & Mahnaz

As no colonies were obtained after one night culture, a new 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.

Concentration of insert and plasmid were determinated again by Nano drop. For each 20μl of reaction, mix the following reagents :

  • 0.65 µL of insert
  • 1.1 µL of plasmid
  • 8,25 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). Two different buffer mix were tested (the usual one and another one from Philippe). 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 containing controls (no buffer mix and plasmid alone) using the usual protocol. As mentioned before, two buffer mix were used. Furthermore, two different DH5a strains were tested (the usual one and another one from Philippe) and at least 12 transformants were cultured overnight : 3 (1 Gibson and 2 controls) x 2 buffer mix x 2 DH5a strains.

Q5 PCR on FKBP in pJET and gblock 2.2 (part of dCas9 NM - GFP 10) for new clonage strategy

By Maxence & Mahnaz

An other clonage strategy was performed by using Gibson between FKBP, gblock 2.2 (part of dCas9 NM - GFP 10) and pSB1C3 (the one from dCas9 ST - GFP 11) in order to have FKBP - GFP 10 in pSB1C3 (pPS16_018). For that purpose, 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)
  • 2.5 µL of each primer mix (10µM)
  • 10 µL of Q5 buffer (5X)
  • 0,5 µL of Q5 high fidelity polymerase
  • 32,5 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 $\infty$

Primers used were:

Matrix gblock FKBP in pJET clones 3, 4 and 5 gblock 2.2 in pUC19
Primers iPS145 and iPS146 iPS147 and iPS84
Tm 62°C 70°C
t 15 sec 15 sec

PCR on GFP 1.9 in pUC19 with 3% DMSO

By Maxence & Mahnaz

As the annealing temperature (Tm) seems too high to obtain good results for GFP 1.9 in pUC19 amplification, DMSO was used in order to reduce the annealing temperature during the PCR. For that purpose, 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)
  • 2.5 µL of each primer mix (10µM)
  • 10 µL of buffer (5X)
  • 0,5 µL of Phusion polymerase
  • 31 µL of nuclease free water
  • 1.5 µL of DMSO

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
60°C 30sec
72°C 30sec
Final Extension 72°C 2min
Hold 4°C $\infty$

Primers used were:

Matrix GFP 1.9 in pUC19
Primers iPS140 and iPS84

PCR Clean-up of PCR products

By Maxence & Mahnaz

PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.

NanoDrop Measurements

By Maxence & Mahnaz

Sample Concentration (ng/µL)
PCR fragment FKBP clone 3
 222.57
PCR fragment FKBP clone 4
 219.99
PCR fragment FKBP clone 5
 376.2
PCR fragment gblock 2.2
 276.33

Gel of cleaned up PCR products

By Maxence & Mahnaz

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 :

PCR products Expected band size (bp)
FKBP 419
gblock 2.2 339
GFP 1.9 862
Result of the migration

All PCR products were at the good size.

Result of the migration

All PCR product were at the good size.

Result of the migration

PCR product was at the good size.





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