Difference between revisions of "Team:Paris Saclay/Notebook/October/14"

(Created page with "{{Team:Paris_Saclay/notebook_header}} = Thursday 14<sup>th</sup> October= ===Visualization=== ====PCR of GFP 1.9 in pUC19 (pPS16_009) and GFP 1.9 in gblock==== ''By Sylvie'...")
 
(→‎Visualization)
Line 55: Line 55:
 
|-
 
|-
 
!Matrix
 
!Matrix
−
!GFP 1.9 in pUC19 (pPS16_009) extracted the 2nd October
+
!GFP 1.9 in pUC19 (pPS16_009) or GFP 1.9 in gblock
 
|-
 
|-
 
|Primers
 
|Primers
Line 61: Line 61:
 
|-
 
|-
 
|}
 
|}
 +
 +
====PCR of 24 extracted GFP 1.9 in pSB1C3 (pPS16_020) from the 13th October====
 +
''By Sylvie''
 +
 +
A PCR was run on 24 extracted plasmids pPS16_020 in order to verify if the clonage worked.
 +
 +
For each 50μl of reaction, mix the following reagents :
 +
* 1 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
 +
* 31.5 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
 +
|95°C
 +
|5min
 +
|-
 +
|rowspan="3"|30 cycles
 +
|95°C
 +
|30sec
 +
|-
 +
|48°C
 +
|30sec
 +
|-
 +
|72°C
 +
|30 sec
 +
|-
 +
|Final Extension
 +
|72°C
 +
|5min
 +
|-
 +
|Hold
 +
|4°C
 +
|$\infty$
 +
|}
 +
 +
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
 +
 +
{| class="wikitable"
 +
|-
 +
!Matrix
 +
!GFP 1.9 in pSB1C3 (pPS16_020) extracted the 13th October
 +
|-
 +
|Primers
 +
|iPS168 and iPS169
 +
|-
 +
|}
 +
 +
====Gel of PCR products====
 +
''By Sylvie''
  
 
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.
 
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.
Line 71: Line 130:
 
!Expected band size (bp)
 
!Expected band size (bp)
 
|-
 
|-
−
|GFP 1.9
+
|GFP 1.9 (iPS84 & iPS140)
 
|862
 
|862
 +
|-
 +
|GFP 1.9 (iPS168 & iPS169)
 +
|1135
 
|-
 
|-
 
|}
 
|}
  
−
[[File:T--Paris Saclay--Gel11111119.png|400px|thumb|center|Result of the migration]]
+
GEL SYLVIE 7
 +
 
 +
No good clones were obtained for GFP 1.9 in pSB1C3 (pPS16_020).
 +
 
 +
====Digestion of FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021), GFP 1.9 PCR product and linearized pSB1C3====
 +
 
 +
Linearized pSB1C3 was digested by restriction enzymes XbaI & PstI:
 +
 
 +
* 4 µL of plasmid
 +
* 1 µL of buffer FD
 +
* 0.5 µL of restriction enzyme XbaI
 +
* 0.5 µL of restriction enzyme PstI
 +
* 2 µL of water
 +
 
 +
GFP 1.9 PCR product was digested by restriction enzymes XbaI & PstI:
 +
 
 +
* 5 µL of GFP 1.9 PCR product
 +
* 1.5 µL of buffer FD
 +
* 1 µL of restriction enzyme XbaI
 +
* 1 µL of restriction enzyme PstI
 +
* 6.5 µL of water
 +
 
 +
FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) was digested by restriction enzymes SpeI & PstI:
 +
 
 +
* 10 µL plasmid
 +
* 2 µL of buffer FD
 +
* 1 µL of restriction enzyme SpeI
 +
* 1 µL of restriction enzyme PstI
 +
* 6 µL of water
 +
 
 +
The mix were incubated for 30 minutes at 37°C.  
 +
 
 +
====Ligation of GFP 1.9 PCR product with PCR blunt====
 +
 
 +
DNA ligase was used to join the sticky ends of the template and vector together:
 +
 
 +
* 1 µL of vector
 +
* 3 µL of GFP 1.9 PCR product
 +
* 2 µL of Buffer T4 10X
 +
* 1 µL of ligase T4 enzyme
 +
* 3 µL of water
 +
 
 +
The mix were incubated for 30 minutes at rooming temperature.
 +
 
 +
====Double transformation of DH5a cells with FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) and GFP 1.9 in pUC19 (pPS16_009)====
 +
''By Sylvie''
 +
 
 +
Dh5a cells were double transformed with pSB1C3 containing FRB - GFP 11 - FKBP - GFP 10 (pPS16_021) and with pUC19 containing GFP 1.9 (pPS16_009) using the usual [[Team:Paris_Saclay/Experiments#heat-shocktransformation|protocol]]. For that purpose, petri dishes used contained LB + Amp + Cm and two concentration of plasmids were used: 1 µL of plasmids not diluted and 1 µL of plasmids diluted 10X.
 +
 
 +
==== Glycerol stocks of clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021)====
 +
''By Sylvie''
 +
 
 +
The glycerol stock of the bacteria with the following plasmids were made.
 +
*pPS16_021 (FRB - GFP 11 - FKBP - GFP 10)

Revision as of 12:41, 18 October 2016

Thursday 14th October

Visualization

PCR of GFP 1.9 in pUC19 (pPS16_009) and GFP 1.9 in gblock

By Sylvie

A new PCR was run in order to obtain a new GFP 1.9 PCR product. For each amplification, two matrix were used: GFP 1.9 in pUC19 (pPS16_009) and GFP 1.9 in gblock.

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

  • 1 µL of matrix (diluted 10X)
  • 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 5min
Hold 4°C $\infty$

Primers used were:

Matrix GFP 1.9 in pUC19 (pPS16_009) or GFP 1.9 in gblock
Primers iPS84 and iPS140

PCR of 24 extracted GFP 1.9 in pSB1C3 (pPS16_020) from the 13th October

By Sylvie

A PCR was run on 24 extracted plasmids pPS16_020 in order to verify if the clonage worked.

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

  • 1 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
  • 31.5 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 95°C 5min
30 cycles 95°C 30sec
48°C 30sec
72°C 30 sec
Final Extension 72°C 5min
Hold 4°C $\infty$

Primers used were:

Matrix GFP 1.9 in pSB1C3 (pPS16_020) extracted the 13th October
Primers iPS168 and iPS169

Gel of PCR products

By Sylvie

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 (iPS84 & iPS140) 862
GFP 1.9 (iPS168 & iPS169) 1135

GEL SYLVIE 7

No good clones were obtained for GFP 1.9 in pSB1C3 (pPS16_020).

Digestion of FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021), GFP 1.9 PCR product and linearized pSB1C3

Linearized pSB1C3 was digested by restriction enzymes XbaI & PstI:

  • 4 µL of plasmid
  • 1 µL of buffer FD
  • 0.5 µL of restriction enzyme XbaI
  • 0.5 µL of restriction enzyme PstI
  • 2 µL of water

GFP 1.9 PCR product was digested by restriction enzymes XbaI & PstI:

  • 5 µL of GFP 1.9 PCR product
  • 1.5 µL of buffer FD
  • 1 µL of restriction enzyme XbaI
  • 1 µL of restriction enzyme PstI
  • 6.5 µL of water

FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) was digested by restriction enzymes SpeI & PstI:

  • 10 µL plasmid
  • 2 µL of buffer FD
  • 1 µL of restriction enzyme SpeI
  • 1 µL of restriction enzyme PstI
  • 6 µL of water

The mix were incubated for 30 minutes at 37°C.

Ligation of GFP 1.9 PCR product with PCR blunt

DNA ligase was used to join the sticky ends of the template and vector together:

  • 1 µL of vector
  • 3 µL of GFP 1.9 PCR product
  • 2 µL of Buffer T4 10X
  • 1 µL of ligase T4 enzyme
  • 3 µL of water

The mix were incubated for 30 minutes at rooming temperature.

Double transformation of DH5a cells with FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) and GFP 1.9 in pUC19 (pPS16_009)

By Sylvie

Dh5a cells were double transformed with pSB1C3 containing FRB - GFP 11 - FKBP - GFP 10 (pPS16_021) and with pUC19 containing GFP 1.9 (pPS16_009) using the usual protocol. For that purpose, petri dishes used contained LB + Amp + Cm and two concentration of plasmids were used: 1 µL of plasmids not diluted and 1 µL of plasmids diluted 10X.

Glycerol stocks of clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021)

By Sylvie

The glycerol stock of the bacteria with the following plasmids were made.

  • pPS16_021 (FRB - GFP 11 - FKBP - GFP 10)