Difference between revisions of "Team:Paris Saclay/Notebook/October/4"

(Created page with "{{Team:Paris_Saclay/notebook_header}} = Tuesday 4<sup>th</sup> October= ==Lab work== ===Visualization=== ====Colony PCR of clones containing FRB - GFP 11 - FKBP - GFP 10 in...")
 
(PCR of extracted GFP 1.9 in pSB1C3 (pPS16_009))
 
(12 intermediate revisions by 2 users not shown)
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= Tuesday 4<sup>th</sup> October=
 
= Tuesday 4<sup>th</sup> October=
==Lab work==
+
 
 
===Visualization===
 
===Visualization===
  
====Colony PCR of clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021)====
+
====Culture of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 and GFP 1.9 in pUC19 (pPS16_009) clone 1====
 
''By Maxence''
 
''By Maxence''
  
For that purpose, X clones 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 no clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) were obtained and as the concentrations of plasmids extracted the 2nd October were too low, the following clones were put on 2 x 10 mL liquid cultures (LB + 30 µg/mL Cm) grown at 37°C overnight:
  
For each clones contained in 20 μl water, 5.13 μL of the following mix were added :
+
*pPS16_018 (FKBP - GFP 10) clone 6
* 2.5 µL DreamTaq Buffer
+
*pPS16_019 (FRB - GFP 11) clone 4
* 0.5 µL of dNTPs (10mM)
+
* 1 µL of each primer mix (10µM)
+
* 0.13 μl of DreamTaq Pol
+
  
PCR was performed as follow:  
+
====PCR of extracted GFP 1.9 in pUC19 (pPS16_009)====
 +
''By Maxence''
 +
 
 +
As no good clones were obtained for GFP 1.9 in pSB1C3 (pPS16_020), a new PCR was run using extracted plasmids containing GFP 1.9 in pUC19 (pPS16_009) extracted the 2nd October. For each amplification, two matrix were used as two liquid cultures were set the 1st October.
 +
 
 +
For each 50μl of reaction, mix the following reagents :
 +
* 2 µL of matrix
 +
* 1 µL of dNTPs (10mM)
 +
* 2.5 µL of each primer mix (10µM)
 +
* 10 µL of buffer (5X)
 +
* 0,5 µL of Phusion polymerase
 +
* 30 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"
 
{| class="wikitable"
 
|-
 
|-
Line 24: Line 36:
 
|-
 
|-
 
|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
 
|-
 
|-
|48.4°C
+
|60°C
|30 sec
+
|30sec
 
|-
 
|-
 
|72°C
 
|72°C
|1 min
+
|30 sec
 
|-
 
|-
 
|Final Extension
 
|Final Extension
 
|72°C
 
|72°C
|7 min
+
|2min
 
|-
 
|-
 
|Hold
 
|Hold
Line 51: Line 63:
 
|-
 
|-
 
!Matrix
 
!Matrix
!Clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021)
+
!GFP 1.9 in pUC19 (pPS16_009) extracted the 2nd October
 
|-
 
|-
 
|Primers
 
|Primers
|iPS168 and iPS169
+
|iPS84 and iPS140
 
|-
 
|-
 
|}
 
|}
  
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.
+
5 µL of PCR products and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity.
  
 
PCR products expected were :
 
PCR products expected were :
Line 67: Line 79:
 
!Expected band size (bp)
 
!Expected band size (bp)
 
|-
 
|-
|FRB - GFP 11 - FKBP - GFP 10
+
|GFP 1.9
|1714
+
|862
 +
|-
 
|}
 
|}
  
GEL
+
[[File:T--Paris Saclay--Gel11111119.png|400px|thumb|center|Result of the migration]]
 +
 
 +
====Cloning of GFP 1.9 PCR product in pSB1C3 by digestion-ligation====
 +
''By Maxence''
 +
 
 +
For that purpose, GFP 1.9 PCR product from today was cut by restriction enzymes PstI & XbaI as following:
 +
 
 +
For the most concentrated one (GFP 1.9 - 1):
 +
 
 +
* 10 µL of GFP 1.9 PCR product
 +
* 4 µL of buffer FD
 +
* 1 µL of restriction enzyme PstI
 +
* 1 µL of restriction enzyme XbaI
 +
* 24 µL of water
 +
 
 +
For the less concentrated one (GFP 1.9 - 2):
 +
 
 +
* 20 µL of GFP 1.9 PCR product
 +
* 4 µL of buffer FD
 +
* 1 µL of restriction enzyme PstI
 +
* 1 µL of restriction enzyme XbaI
 +
* 14 µL of water
 +
 
 +
Furthermore, BbaB0015 plasmid (in order to have pSB1C3) was cut by restriction enzymes PstI & XbaI and was treated by the alkaline phosphatase FastAP (dephosphorylation of cloning vector DNA to prevent recircularization during ligation)  as following:
 +
 
 +
* 20 µL of BbaB0015 plasmid
 +
* 4 µL of buffer FD
 +
* 1 µL of restriction enzyme EcoRI
 +
* 1 µL of restriction enzyme XbaI
 +
* 1 µL of alkaline phosphatase FastAP
 +
* 13 µL of water
 +
 
 +
The mix were incubated for 1 hour at 37°C. Then, 5 µL of each digestion products and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
 +
 
 +
Migration products expected were :
 +
 
 +
{| class="wikitable"
 +
|-
 +
!Migration products
 +
!Expected band size (bp)
 +
|-
 +
|Template digested (GFP 1.9 PCR product treated by PstI & XbaI)
 +
|900
 +
|-
 +
|Vector digested (BbaB0015 treated by PstI & XbaI)
 +
|2000
 +
|-
 +
|}
 +
 
 +
[[File:T--Paris Saclay--Gel111111110.png|400px|thumb|center|Result of the migration]]
 +
 
 +
The digestion products were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]]. After cleaned-up, sample from extract 1 and sample from extract 2 were pooled together. Then 2 µL of each cleaned-up products and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
 +
 
 +
[[File:T--Paris Saclay--Gel111111111.png|400px|thumb|center|Result of the migration]]
 +
 
 +
Once template and vector were cut and purified, DNA ligase was used to join the sticky ends of the template and vector together:
 +
 
 +
* 9 µL of template (GFP 1.9 PCR product treated by PstI & XbaI)
 +
* 3 µL of vector (BbaB0015 treated by PstI & XbaI)
 +
* 2 µL of Buffer T4 10X
 +
* 1 µL of ligase T4 enzyme
 +
* 5 µL of water
 +
 
 +
A control was done without template. The mix were incubated for 1 hour at rooming temperature. Then the ligase was desactivated at 75°C for 10 minutes.
 +
 
 +
====Transformation of DH5a cells with and GFP 1.9 in pSB1C3 (pPS16_020) obtained by Digestion-Ligation====
 +
''By Maxence''
 +
 
 +
Dh5a cells were transformed with pSB1C3 containing GFP 1.9 (pPS16_020) or controls (digested plasmid) using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]].
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 12:09, 18 October 2016

Tuesday 4th October

Visualization

Culture of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 and GFP 1.9 in pUC19 (pPS16_009) clone 1

By Maxence

As no clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) were obtained and as the concentrations of plasmids extracted the 2nd October were too low, the following clones were put on 2 x 10 mL liquid cultures (LB + 30 µg/mL Cm) grown at 37°C overnight:

  • pPS16_018 (FKBP - GFP 10) clone 6
  • pPS16_019 (FRB - GFP 11) clone 4

PCR of extracted GFP 1.9 in pUC19 (pPS16_009)

By Maxence

As no good clones were obtained for GFP 1.9 in pSB1C3 (pPS16_020), a new PCR was run using extracted plasmids containing GFP 1.9 in pUC19 (pPS16_009) extracted the 2nd October. For each amplification, two matrix were used as two liquid cultures were set the 1st October.

For each 50μl of reaction, mix the following reagents :

  • 2 µL of matrix
  • 1 µL of dNTPs (10mM)
  • 2.5 µL of each primer mix (10µM)
  • 10 µL of buffer (5X)
  • 0,5 µL of Phusion polymerase
  • 30 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 30 sec
Final Extension 72°C 2min
Hold 4°C $\infty$

Primers used were:

Matrix GFP 1.9 in pUC19 (pPS16_009) extracted the 2nd October
Primers iPS84 and iPS140

5 µL of PCR products and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity.

PCR products expected were :

PCR products Expected band size (bp)
GFP 1.9 862
Result of the migration

Cloning of GFP 1.9 PCR product in pSB1C3 by digestion-ligation

By Maxence

For that purpose, GFP 1.9 PCR product from today was cut by restriction enzymes PstI & XbaI as following:

For the most concentrated one (GFP 1.9 - 1):

  • 10 µL of GFP 1.9 PCR product
  • 4 µL of buffer FD
  • 1 µL of restriction enzyme PstI
  • 1 µL of restriction enzyme XbaI
  • 24 µL of water

For the less concentrated one (GFP 1.9 - 2):

  • 20 µL of GFP 1.9 PCR product
  • 4 µL of buffer FD
  • 1 µL of restriction enzyme PstI
  • 1 µL of restriction enzyme XbaI
  • 14 µL of water

Furthermore, BbaB0015 plasmid (in order to have pSB1C3) was cut by restriction enzymes PstI & XbaI and was treated by the alkaline phosphatase FastAP (dephosphorylation of cloning vector DNA to prevent recircularization during ligation) as following:

  • 20 µL of BbaB0015 plasmid
  • 4 µL of buffer FD
  • 1 µL of restriction enzyme EcoRI
  • 1 µL of restriction enzyme XbaI
  • 1 µL of alkaline phosphatase FastAP
  • 13 µL of water

The mix were incubated for 1 hour at 37°C. Then, 5 µL of each digestion products and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

Migration products expected were :

Migration products Expected band size (bp)
Template digested (GFP 1.9 PCR product treated by PstI & XbaI) 900
Vector digested (BbaB0015 treated by PstI & XbaI) 2000
Result of the migration

The digestion products were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol. After cleaned-up, sample from extract 1 and sample from extract 2 were pooled together. Then 2 µL of each cleaned-up products and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

Result of the migration

Once template and vector were cut and purified, DNA ligase was used to join the sticky ends of the template and vector together:

  • 9 µL of template (GFP 1.9 PCR product treated by PstI & XbaI)
  • 3 µL of vector (BbaB0015 treated by PstI & XbaI)
  • 2 µL of Buffer T4 10X
  • 1 µL of ligase T4 enzyme
  • 5 µL of water

A control was done without template. The mix were incubated for 1 hour at rooming temperature. Then the ligase was desactivated at 75°C for 10 minutes.

Transformation of DH5a cells with and GFP 1.9 in pSB1C3 (pPS16_020) obtained by Digestion-Ligation

By Maxence

Dh5a cells were transformed with pSB1C3 containing GFP 1.9 (pPS16_020) or controls (digested plasmid) using the usual protocol.