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

(→‎PCR on FKBP in pJET and gblock 2.2 (part of dCas9 NM - GFP 10) for new clonage strategy)
(→‎Thursday 8th September)
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====Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3====
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====PCR on GFP 1.9 in pUC19 with 3% DMSO ====
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''Maxence''
+
''By 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.
+
As the annealing temperature (Tm) seems to 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 clones contained in 20 μl water, 4.13 μL of the following mix were added :
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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)
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* 2.5 µL of each primer mix (10µM)
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* 0.13 μl of DreamTaq Pol
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* 10 µL of buffer (5X)
 +
* 0,5 µL of Phusion polymerase
 +
* 31 µL of nuclease free water
 +
* 1.5 µL of DMSO
  
−
PCR was performed as follow:  
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Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice.
 +
Perform PCR as follow:  
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
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|-
 
|-
 
|Initial denaturation
 
|Initial denaturation
−
|95°C
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|98°C
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|3 min
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|30sec
 
|-
 
|-
 
|rowspan="3"|30 cycles
 
|rowspan="3"|30 cycles
−
|95°C
+
|98°C
−
|30 sec
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|10sec
 
|-
 
|-
 
|Tm
 
|Tm
−
|30 sec
+
|30sec
 
|-
 
|-
 
|72°C
 
|72°C
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|Final Extension
 
|Final Extension
 
|72°C
 
|72°C
−
|7 min
+
|2min
 
|-
 
|-
 
|Hold
 
|Hold
 
|4°C
 
|4°C
−
|$\infinity\$
+
|$\infty$
 
|}
 
|}
  
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|-
 
|-
 
!Matrix
 
!Matrix
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!Clones containing dCas9 NM - GFP 10 in pSB1C3
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!gblock 2.2 in pUC19
 
|-
 
|-
 
|Primers
 
|Primers
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|iPS83 and iPS84
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|iPS140 and iPS84
 
|-
 
|-
 
|Tm
 
|Tm
−
|63.6°C
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|60°C
 
|-
 
|-
 
|t
 
|t
−
|1 min 30
+
|30 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 Clean-up of PCR products ====
 +
''By Maxence''
 +
 
 +
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]].
 +
 
 +
====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 :
 
PCR products expected were :
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!Expected band size (bp)
 
!Expected band size (bp)
 
|-
 
|-
−
|dCas9 NM - GFP 10 - pSB1C3
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|FKBP
−
|3688
+
|X
 +
|-
 +
|gblock 2.2
 +
|X
 +
|-
 +
|GFP 1.9
 +
|862
 
|}
 
|}
  
−
[[File:T--Paris Saclay--160826 gel ligation fragm1 et 2.jpg.png|400px|thumb|center|Result of the migration]]
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GEL 4 : FKBP
 +
 
 +
All PCR products were at the good size.  
  
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GEL GEL GEL
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GEL 5 : gblock 2.2
  
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====Samples preparation for sequencing====
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The PCR product was at the good size.
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''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.
+
GEL 6 : gfp 1.9

Revision as of 17:41, 13 September 2016

Thursday 8th September

Lab work

Visualization

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

By Maxence

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 obtained by Gibson

By Maxence

Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11, 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

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.

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

As the annealing temperature (Tm) seems to 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
Tm 30sec
72°C t
Final Extension 72°C 2min
Hold 4°C $\infty$ 
Primers used were:
Matrix gblock 2.2 in pUC19
Primers iPS140 and iPS84
Tm 60°C
t 30 sec

PCR Clean-up of PCR products

By Maxence

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

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 :

PCR products Expected band size (bp)
FKBP X
gblock 2.2 X
GFP 1.9 862

GEL 4 : FKBP

All PCR products were at the good size.

GEL 5 : gblock 2.2

The PCR product was at the good size.

GEL 6 : gfp 1.9