Difference between revisions of "Team:Paris Saclay/Notebook/September/22"

(→‎Thuersday 22nd September)
(→‎Lab work)
 
(19 intermediate revisions by 2 users not shown)
Line 1: Line 1:
 
{{Team:Paris_Saclay/notebook_header}}
 
{{Team:Paris_Saclay/notebook_header}}
  
−
= Thuersday 22<sup>nd</sup> September=
+
=Thursday 22<sup>nd</sup> September=
−
==Lab work==
+
 
 
===Visualization===
 
===Visualization===
  
−
==== Glycerol stocks of clones X, X, X and X containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)====
+
====Colony PCR of 8 clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)====
−
"By Maxence, Mahnaz & Coline"
+
''By Maxence, Mahnaz & Coline''
  
−
The glycerol stock of the bacteria with the following plasmids were made.
+
A colony PCR was done for 8 clones (4 clones with GFP 1.9 PCR product obtained with DMSO and 4 clones with GFP 1.9 PCR product obtained without DMSO) from the 20th September. For that purpose, 8 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.
−
*pPS16_017 (dCas9 ST - GFP 11) clone X
+
−
*pPS16_017 (dCas9 ST - GFP 11) clone X
+
−
*pPS16_017 (dCas9 ST - GFP 11) clone X
+
−
*pPS16_017 (dCas9 ST - GFP 11) clone X
+
  
−
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
+
For each clones contained in 20 μl water, 5.13 μL of the following mix were added :
 +
* 2.5 µL DreamTaq Buffer
 +
* 0.5 µL of dNTPs (10mM)
 +
* 1 µL of each primer mix (10µM)
 +
* 0.13 μl of DreamTaq Pol
  
−
====Plasmids extraction of clones X, X, X and X containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)====
+
PCR was performed as follow:
−
"By Maxence, Mahnaz & Coline"
+
{| class="wikitable"
 +
|-
 +
!Step
 +
!Temperature
 +
!Time
 +
|-
 +
|Initial denaturation
 +
|95°C
 +
|3 min
 +
|-
 +
|rowspan="3"|30 cycles
 +
|95°C
 +
|30 sec
 +
|-
 +
|48.4°C
 +
|30 sec
 +
|-
 +
|72°C
 +
|1min 30sec
 +
|-
 +
|Final Extension
 +
|72°C
 +
|7 min
 +
|-
 +
|Hold
 +
|4°C
 +
|$\infty$
 +
|}
  
−
The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit:
+
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
−
*pPS16_017 (dCas9 ST - GFP 11) clone X
+
−
*pPS16_017 (dCas9 ST - GFP 11) clone X
+
−
*pPS16_017 (dCas9 ST - GFP 11) clone X
+
−
*pPS16_017 (dCas9 ST - GFP 11) clone X
+
  
 +
{| class="wikitable"
 +
|-
 +
!Matrix
 +
!Clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
 +
|-
 +
|Primers
 +
|iPS168 and iPS169
 +
|-
 +
|}
  
−
====Digestion of plasmids extracted from clones X, X, X and X containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)====
+
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.
−
"By Maxence, Mahnaz & Coline"
+
  
−
To assess if DH5a cells were transformed with the good plasmid, plasmid digestion was run with XbaI as following:  
+
PCR products expected were :
  
−
* 1 µL of extracted plasmid
+
{| class="wikitable"
−
* 1 µL of buffer
+
|-
−
* 1 µL of restriction enzyme XbaI
+
!PCR products
−
* 7 µL of water
+
!Expected band size (bp)
 +
|-
 +
|FRB - GFP 11 - GFP 1.9
 +
|1800
 +
|}
  
−
A control was done with 1 µL of non-digested plasmid.
+
[[File:T--Paris Saclay--Gel111112.png|400px|thumb|center|Result of the migration]]
  
−
GEL
+
PCR products were not at the good size.
  
−
====Colony PCR of 16 clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)====
+
====Colony PCR of 12 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_019)====
 
''By Maxence, Mahnaz & Coline''
 
''By Maxence, Mahnaz & Coline''
  
−
A colony PCR was done for 16 clones from the 20th September. For that purpose, 16 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 sequencing results were not convincing, a new colony PCR was done for 12 clones from the 15th September (8 new clones and the 4 sent for sequencing) but with primers iPS168 & iPS169. For that purpose, 12 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.
  
 
For each clones contained in 20 μl water, 5.13 μL of the following mix were added :
 
For each clones contained in 20 μl water, 5.13 μL of the following mix were added :
Line 70: Line 105:
 
|-
 
|-
 
|72°C
 
|72°C
−
|1min 30sec
+
|30 sec
 
|-
 
|-
 
|Final Extension
 
|Final Extension
Line 81: Line 116:
 
|}
 
|}
  
−
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
+
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
 +
 
 +
{| class="wikitable"
 +
|-
 +
!Matrix
 +
!Clones containing FKBP - GFP 10 in pSB1C3 (pPS16_19)
 +
|-
 +
|Primers
 +
|iPS83 and iPS84
 +
|-
 +
|}
 +
 
 +
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 products expected were :
 +
 
 +
{| class="wikitable"
 +
|-
 +
!PCR products
 +
!Expected band size (bp)
 +
|-
 +
|FKBP - GFP 10 in pSB1C3 (pPS16_019)
 +
|757
 +
|}
 +
 
 +
[[File:T--Paris Saclay--Gel111113.png|400px|thumb|center|Result of the migration]]
 +
 
 +
PCR products were not at the good size, plasmids were empty.
 +
 
 +
====PCR on cleaned up PCR products FKBP from 8th September and gblock 2.2 from the 8th September in order to fuse fuse these fragments====
 +
''By Maxence, Manhaz & Coline''
 +
 
 +
As sequencing and colony PCR of clones containing FKBP - GFP 10 in pSB1C3 did not show good results, a new clonage strategy was performed: before the Gibson, the two inserts were fused together by PCR. So PCR was performed with cleaned up PCR products FKBP (obtained the 8th September) and claned up PCR product gblock 2.2 (obtained the 8th September) with the following protocol.
 +
 
 +
For each 50μl of reaction, mix the following reagents :
 +
* 1 µL of each PCR product
 +
* 1 µL of dNTPs (10mM)
 +
* 2 µL of each primer mix (10µM)
 +
* 10 µL of buffer (5X)
 +
* 0,5 µL of Phusion 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:
 +
{| class="wikitable"
 +
|-
 +
!Step
 +
!Temperature
 +
!Time
 +
|-
 +
|Initial denaturation
 +
|98°C
 +
|30sec
 +
|-
 +
|rowspan="3"|30 cycles
 +
|98°C
 +
|10sec
 +
|-
 +
|65°C
 +
|30sec
 +
|-
 +
|72°C
 +
|20sec
 +
|-
 +
|Final Extension
 +
|72°C
 +
|7min
 +
|-
 +
|Hold
 +
|4°C
 +
|$\infty$
 +
|}
 +
 
 +
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
 +
 
 +
{| class="wikitable"
 +
|-
 +
!Matrix
 +
!PCR products FKBP from 8th September and gblock 2.2 from the 8th September
 +
|-
 +
|Primers
 +
|iPS145 and iPS84
 +
|-
 +
|}
 +
 
 +
====Gel of PCR products====
 +
''By Maxence, Mahnaz & Coline''
 +
 
 +
After amplification, the totality of PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
 +
 
 +
PCR products expected were :
 +
 
 +
{| class="wikitable"
 +
|-
 +
!PCR products
 +
!Expected band size (bp)
 +
|-
 +
|FKBP - GFP 10
 +
|747
 +
|}
 +
 
 +
[[File:T--Paris Saclay--Gel111114.png|400px|thumb|center|Result of the migration]]
 +
 
 +
PCR product was at the good size.
 +
 
 +
====Clean-up of PCR products from gel====
 +
''By Maxence, Mahnaz & Coline''
 +
 
 +
The PCR products were cleaned up from the gel by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]]. Their concentrations were assessed by NanoDrop:
 +
 
 +
====Colony PCR of 8 clones containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)====
 +
''By Maxence, Mahnaz & Coline''
 +
 
 +
A colony PCR was done for 8 clones from the 20th September. For that purpose, 8 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.
 +
 
 +
For each clones contained in 20 μl water, 5.13 μL of the following mix were added :
 +
* 2.5 µL DreamTaq Buffer
 +
* 0.5 µL of dNTPs (10mM)
 +
* 1 µL of each primer mix (10µM)
 +
* 0.13 μl of DreamTaq Pol
 +
 
 +
PCR was performed as follow:
 +
{| class="wikitable"
 +
|-
 +
!Step
 +
!Temperature
 +
!Time
 +
|-
 +
|Initial denaturation
 +
|95°C
 +
|3 min
 +
|-
 +
|rowspan="3"|25 cycles
 +
|95°C
 +
|30 sec
 +
|-
 +
|48.4°C
 +
|30 sec
 +
|-
 +
|72°C
 +
|4min
 +
|-
 +
|Final Extension
 +
|72°C
 +
|7 min
 +
|-
 +
|Hold
 +
|4°C
 +
|$\infty$
 +
|}
 +
 
 +
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
  
 
{| class="wikitable"
 
{| class="wikitable"
Line 92: Line 278:
 
|-
 
|-
 
|}
 
|}
 +
 +
====Colony PCR of 8 clones containing GFP 1.9 in pSB1C3 (pPS16_020)====
 +
''By Maxence, Mahnaz & Coline''
 +
 +
As sequencing results were not good, a new colony PCR was done for 8 clones from the 12th September. For that purpose, 8 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.
 +
 +
For each clones contained in 20 μl water, 5.13 μL of the following mix were added :
 +
* 2.5 µL DreamTaq Buffer
 +
* 0.5 µL of dNTPs (10mM)
 +
* 1 µL of each primer mix (10µM)
 +
* 0.13 μl of DreamTaq Pol
 +
 +
PCR was performed as follow:
 +
{| class="wikitable"
 +
|-
 +
!Step
 +
!Temperature
 +
!Time
 +
|-
 +
|Initial denaturation
 +
|95°C
 +
|3 min
 +
|-
 +
|rowspan="3"|30 cycles
 +
|95°C
 +
|30 sec
 +
|-
 +
|48.4
 +
|30 sec
 +
|-
 +
|72°C
 +
|30sec
 +
|-
 +
|Final Extension
 +
|72°C
 +
|7 min
 +
|-
 +
|Hold
 +
|4°C
 +
|$\infty$
 +
|}
 +
 +
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
 +
 +
{| class="wikitable"
 +
|-
 +
!Matrix
 +
!Clones containing GFP 1.9 in pSB1C3 (pPS16_020)
 +
|-
 +
|Primers
 +
|iPS168 and iPS169
 +
|-
 +
|}
 +
 +
====Gel of PCR products====
 +
''By Maxence, Mahnaz & Coline''
  
 
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.
 
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.
Line 102: Line 344:
 
!Expected band size (bp)
 
!Expected band size (bp)
 
|-
 
|-
−
|FRB - GFP 11 - GFP 1.9
+
|dCas9 ST - GFP 11
−
|1500
+
|3800
 +
|-
 +
|GFP 1.9 in pSB1C3
 +
|1135
 
|}
 
|}
  
−
GEL
+
[[File:T--Paris Saclay--Gel111115.png|400px|thumb|center|Result of the migration]]
 +
 
 +
For GFP 1.9 in pSB1C3, all PCR products were at the good size, clones 3, 4, 7 and 8 were selected for sequencing. For dCas ST - GFP 11, 6 PCR products were at the good size, clones 2, 7 and 8 were selected for sequencing.
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 17:08, 9 October 2016

Thursday 22nd September

Visualization

Colony PCR of 8 clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)

By Maxence, Mahnaz & Coline

A colony PCR was done for 8 clones (4 clones with GFP 1.9 PCR product obtained with DMSO and 4 clones with GFP 1.9 PCR product obtained without DMSO) from the 20th September. For that purpose, 8 clones were screened and used for the usual 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.

For each clones contained in 20 μl water, 5.13 μL of the following mix were added :

  • 2.5 µL DreamTaq Buffer
  • 0.5 µL of dNTPs (10mM)
  • 1 µL of each primer mix (10µM)
  • 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step Temperature Time
Initial denaturation 95°C 3 min
30 cycles 95°C 30 sec
48.4°C 30 sec
72°C 1min 30sec
Final Extension 72°C 7 min
Hold 4°C $\infty$

Primers used were:

Matrix Clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
Primers iPS168 and iPS169

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 products expected were :

PCR products Expected band size (bp)
FRB - GFP 11 - GFP 1.9 1800
Result of the migration

PCR products were not at the good size.

Colony PCR of 12 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_019)

By Maxence, Mahnaz & Coline

As sequencing results were not convincing, a new colony PCR was done for 12 clones from the 15th September (8 new clones and the 4 sent for sequencing) but with primers iPS168 & iPS169. For that purpose, 12 were screened and used for the usual 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.

For each clones contained in 20 μl water, 5.13 μL of the following mix were added :

  • 2.5 µL DreamTaq Buffer
  • 0.5 µL of dNTPs (10mM)
  • 1 µL of each primer mix (10µM)
  • 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step Temperature Time
Initial denaturation 95°C 3 min
30 cycles 95°C 30 sec
48.4°C 30 sec
72°C 30 sec
Final Extension 72°C 7 min
Hold 4°C $\infty$

Primers used were:

Matrix Clones containing FKBP - GFP 10 in pSB1C3 (pPS16_19)
Primers iPS83 and iPS84

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 products expected were :

PCR products Expected band size (bp)
FKBP - GFP 10 in pSB1C3 (pPS16_019) 757
Result of the migration

PCR products were not at the good size, plasmids were empty.

PCR on cleaned up PCR products FKBP from 8th September and gblock 2.2 from the 8th September in order to fuse fuse these fragments

By Maxence, Manhaz & Coline

As sequencing and colony PCR of clones containing FKBP - GFP 10 in pSB1C3 did not show good results, a new clonage strategy was performed: before the Gibson, the two inserts were fused together by PCR. So PCR was performed with cleaned up PCR products FKBP (obtained the 8th September) and claned up PCR product gblock 2.2 (obtained the 8th September) with the following protocol.

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

  • 1 µL of each PCR product
  • 1 µL of dNTPs (10mM)
  • 2 µL of each primer mix (10µM)
  • 10 µL of buffer (5X)
  • 0,5 µL of Phusion 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
65°C 30sec
72°C 20sec
Final Extension 72°C 7min
Hold 4°C $\infty$

Primers used were:

Matrix PCR products FKBP from 8th September and gblock 2.2 from the 8th September
Primers iPS145 and iPS84

Gel of PCR products

By Maxence, Mahnaz & Coline

After amplification, the totality of 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 - GFP 10 747
Result of the migration

PCR product was at the good size.

Clean-up of PCR products from gel

By Maxence, Mahnaz & Coline

The PCR products were cleaned up from the gel by using the NucleoSpin Gel and PCR Clean-up kit protocol. Their concentrations were assessed by NanoDrop:

Colony PCR of 8 clones containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)

By Maxence, Mahnaz & Coline

A colony PCR was done for 8 clones from the 20th September. For that purpose, 8 clones were screened and used for the usual 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.

For each clones contained in 20 μl water, 5.13 μL of the following mix were added :

  • 2.5 µL DreamTaq Buffer
  • 0.5 µL of dNTPs (10mM)
  • 1 µL of each primer mix (10µM)
  • 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step Temperature Time
Initial denaturation 95°C 3 min
25 cycles 95°C 30 sec
48.4°C 30 sec
72°C 4min
Final Extension 72°C 7 min
Hold 4°C $\infty$

Primers used were:

Matrix Clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
Primers iPS168 and iPS169

Colony PCR of 8 clones containing GFP 1.9 in pSB1C3 (pPS16_020)

By Maxence, Mahnaz & Coline

As sequencing results were not good, a new colony PCR was done for 8 clones from the 12th September. For that purpose, 8 clones were screened and used for the usual 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.

For each clones contained in 20 μl water, 5.13 μL of the following mix were added :

  • 2.5 µL DreamTaq Buffer
  • 0.5 µL of dNTPs (10mM)
  • 1 µL of each primer mix (10µM)
  • 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step Temperature Time
Initial denaturation 95°C 3 min
30 cycles 95°C 30 sec
48.4 30 sec
72°C 30sec
Final Extension 72°C 7 min
Hold 4°C $\infty$

Primers used were:

Matrix Clones containing GFP 1.9 in pSB1C3 (pPS16_020)
Primers iPS168 and iPS169

Gel of PCR products

By Maxence, Mahnaz & Coline

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 products expected were :

PCR products Expected band size (bp)
dCas9 ST - GFP 11 3800
GFP 1.9 in pSB1C3 1135
Result of the migration

For GFP 1.9 in pSB1C3, all PCR products were at the good size, clones 3, 4, 7 and 8 were selected for sequencing. For dCas ST - GFP 11, 6 PCR products were at the good size, clones 2, 7 and 8 were selected for sequencing.





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