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

(→‎PCR on GFP 1.9 in pUC19 with 3% DMSO)
(→‎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 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.
+
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.
  
 
====Q5 PCR on FKBP in pJET and gblock 2.2 (part of dCas9 NM - GFP 10) for new clonage strategy ====
 
====Q5 PCR on FKBP in pJET and gblock 2.2 (part of dCas9 NM - GFP 10) for new clonage strategy ====
−
''By Maxence''
+
''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. For that purpose, Q5 PCR was performed on plasmids with the following protocol.
+
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 :
 
For each 50μl of reaction, mix the following reagents :
Line 65: Line 67:
 
|}
 
|}
  
−
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
+
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
  
 
{| class="wikitable"
 
{| class="wikitable"
Line 87: Line 89:
  
 
====PCR on GFP 1.9 in pUC19 with 3% DMSO ====
 
====PCR on GFP 1.9 in pUC19 with 3% DMSO ====
−
''By Maxence''
+
''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.
 
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.
Line 116: Line 118:
 
|10sec
 
|10sec
 
|-
 
|-
−
|Tm
+
|60°C
 
|30sec
 
|30sec
 
|-
 
|-
 
|72°C
 
|72°C
−
|t
+
|30sec
 
|-
 
|-
 
|Final Extension
 
|Final Extension
Line 131: Line 133:
 
|}
 
|}
  
−
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
+
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
  
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
 
!Matrix
 
!Matrix
−
!gblock 2.2 in pUC19
+
!GFP 1.9 in pUC19
 
|-
 
|-
 
|Primers
 
|Primers
 
|iPS140 and iPS84
 
|iPS140 and iPS84
 
|-
 
|-
−
|Tm
 
−
|60°C
 
−
|-
 
−
|t
 
−
|30 sec
 
 
|}
 
|}
  
 
====PCR Clean-up of PCR products ====
 
====PCR Clean-up of PCR products ====
−
''By Maxence''
+
''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]].
 
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]].
  
 
====NanoDrop Measurements====
 
====NanoDrop Measurements====
−
''By Maxence''
+
''By Maxence & Mahnaz''
  
 
{| class="wikitable"
 
{| class="wikitable"
Line 175: Line 172:
  
 
====Gel of cleaned up PCR products====
 
====Gel of cleaned up PCR products====
−
''By Maxence''
+
''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.
 
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.
Line 187: Line 184:
 
|-
 
|-
 
|FKBP
 
|FKBP
−
|X
+
|419
 
|-
 
|-
 
|gblock 2.2
 
|gblock 2.2
−
|X
+
|339
 
|-
 
|-
 
|GFP 1.9
 
|GFP 1.9
Line 196: Line 193:
 
|}
 
|}
  
−
GEL 4 : FKBP
+
[[File:T--Paris Saclay--Gel114.png|400px|thumb|center|Result of the migration]]
  
 
All PCR products were at the good size.  
 
All PCR products were at the good size.  
  
−
GEL 5 : gblock 2.2
+
[[File:T--Paris Saclay--Gel115.png|400px|thumb|center|Result of the migration]]
 +
 
 +
All PCR product were at the good size.
 +
 
 +
[[File:T--Paris Saclay--Gel116.png|400px|thumb|center|Result of the migration]]
  
−
The PCR product was at the good size.  
+
PCR product was at the good size.
  
−
GEL 6 : gfp 1.9
+
{{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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