Difference between revisions of "Team:Paris Saclay/Notebook/September/9"

(→‎Friday 9th September)
(→‎Lab work)
 
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 +
{{Team:Paris_Saclay/notebook_header}}
 +
 
= Friday 9<sup>th</sup> September=
 
= Friday 9<sup>th</sup> September=
−
==Lab work==
+
 
 
===Visualization===
 
===Visualization===
  
−
====Colony PCR of 16 clones containing FRB - GFP 11 in pSB1C3====
+
====Colony PCR of 16 clones containing FRB - GFP 11 in pSB1C3 (pPS16_019)====
−
''Maxence''
+
''By Maxence & Mahnaz''
  
 
Colonies were obtained for the Gibson done the 7th September but not for the one done the 8th September. A colony PCR was done for 16 clones from the 7th September. For that purpose, 16 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.
 
Colonies were obtained for the Gibson done the 7th September but not for the one done the 8th September. A colony PCR was done for 16 clones from the 7th September. For that purpose, 16 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.
Line 29: Line 31:
 
|30 sec
 
|30 sec
 
|-
 
|-
−
|Tm
+
|61°C
 
|30 sec
 
|30 sec
 
|-
 
|-
 
|72°C
 
|72°C
−
|t
+
|1 min 30 sec
 
|-
 
|-
 
|Final Extension
 
|Final Extension
Line 44: Line 46:
 
|}
 
|}
  
−
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
+
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
  
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
 
!Matrix
 
!Matrix
−
!Clones containing FRB - GFP 11 in pSB1C3
+
!Clones containing FRB - GFP 11 in pSB1C3 (pPS16_019)
 
|-
 
|-
 
|Primers
 
|Primers
 
|iPS83 and iPS84
 
|iPS83 and iPS84
 
|-
 
|-
−
|Tm
 
−
|61°C
 
−
|-
 
−
|t
 
−
|1 min 30
 
 
|}
 
|}
  
Line 74: Line 71:
 
|}
 
|}
  
−
GEL 6
+
[[File:T--Paris Saclay--Gel117.png|400px|thumb|center|Result of the migration]]
  
−
All PCR products were at the good size and seemed to be concentrated. It could be explained as these clones were grown for 2 days. Clones 7, 9, 15 and 16 were selected and were grown at 37°C overnight.  
+
All PCR products were at the good size and seemed to be concentrated. It could be explained as these clones were grown for 2 days. Clones 4, 9, 13 and 16 were selected and were grown at 37°C overnight.
  
 
====PCR on GFP 1.9 in pUC19, gblock 1.1, gblock 1.2, gblock 2.1 and gblock 2.2 with 3% DMSO ====
 
====PCR on GFP 1.9 in pUC19, gblock 1.1, gblock 1.2, gblock 2.1 and gblock 2.2 with 3% DMSO ====
−
''By Maxence''
+
''By Maxence & Mahnaz''
  
−
As the annealing temperature (Tm) seems too high to obtain good results for these amplifications, 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 these amplifications, 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 amplification, two matrix were used: one was a previous PCR products and one was a plasmid.
  
 
For each 50μl of reaction, mix the following reagents :
 
For each 50μl of reaction, mix the following reagents :
−
* 1 µL of plasmid
+
* 1 µL of matrix
 
* 1 µL of dNTPs (10mM)
 
* 1 µL of dNTPs (10mM)
 
* 2.5 µL of each primer mix (10µM)
 
* 2.5 µL of each primer mix (10µM)
Line 108: Line 105:
 
|10sec
 
|10sec
 
|-
 
|-
−
|Tm
+
|60°C
 
|30sec
 
|30sec
 
|-
 
|-
 
|72°C
 
|72°C
−
|t
+
|30 sec
 
|-
 
|-
 
|Final Extension
 
|Final Extension
Line 123: Line 120:
 
|}
 
|}
  
−
[[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
+
!PCR product of gblock 1.1 from 2nd September (Q5)
 +
!gblock 1.1 9/08
 +
!PCR product of gblock 1.2 from 2nd September (Q5)
 +
!gblock 1.2 11
 +
!PCR product of gblock 2.1 from 2nd September (Q5)
 +
!gblock 2.1 PPS16003
 +
!PCR product of gblock 2.2 from 2nd September (Q5)
 +
!gblock 2.2 PPS16004
 +
!PCR product of GFP 1.9 from 8th September
 +
!GFP 1.9 in pUC19
 
|-
 
|-
 
|Primers
 
|Primers
−
|iPS140 and iPS84
+
|iPS140 and iPS120
−
|-
+
|iPS140 and iPS120
−
|Tm
+
|iPS121 and iPS122
−
|60°C
+
|iPS121 and iPS122
−
|-
+
|iPS123 and iPS124
−
|t
+
|iPS123 and iPS124
−
|30 sec
+
|iPS125 and iPS84
−
|}
+
|iPS125 and iPS84
−
 
+
|iPS84 and iPS140
−
====PCR Clean-up of PCR products ====
+
|iPS84 and iPS140
−
''By Maxence''
+
−
 
+
−
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]].
+
−
 
+
−
====NanoDrop Measurements====
+
−
''By Maxence''
+
−
 
+
−
{| class="wikitable"
+
−
!Sample
+
−
!Concentration (ng/µL)
+
−
|-
+
−
|PCR fragment FKBP clone 3<div id="FKBP clone 3"></div>
+
−
|222.57
+
−
|-
+
−
|PCR fragment FKBP clone 4<div id="FKBP clone 4"></div>
+
−
|219.99
+
−
|-
+
−
|PCR fragment FKBP clone 5<div id="FKBP clone 5"></div>
+
−
|376.2
+
−
|-
+
−
|PCR fragment gblock 2.2<div id="gblock 2.2"></div>
+
−
|276.33
+
 
|-
 
|-
 
|}
 
|}
  
 
====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 178: Line 162:
 
!Expected band size (bp)
 
!Expected band size (bp)
 
|-
 
|-
−
|FKBP
+
|gblock 1.1
−
|X
+
|960
 +
|-
 +
|gblock 1.2
 +
|960
 +
|-
 +
|gblock 2.1
 +
|1023
 
|-
 
|-
 
|gblock 2.2
 
|gblock 2.2
−
|X
+
|808
 
|-
 
|-
 
|GFP 1.9
 
|GFP 1.9
Line 188: Line 178:
 
|}
 
|}
  
−
GEL 4 : FKBP
+
[[File:T--Paris Saclay--Gel118.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
+
====PCR Clean-up of PCR products ====
 +
''By Maxence & Mahnaz''
  
−
The PCR product was at the good size.  
+
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit [[Team:Paris_Saclay/Experiments#Purification|protocol]]. For each amplification, only PCR products from plasmids were cleaned in order to reduce the risks of mutations. For GFP 1.9 amplification products, the 4 samples were pooled before clean up.
  
−
GEL 6 : gfp 1.9
+
====Results about FRB - GFP 11 transformed colonies  from 8th September====
 +
''By Maxence & Mahnaz''
 +
 
 +
During the afternoon, observations were made about the 8th September Gibson, but as results were obtained for the 7th September Gibson, a colony PCR was not run. Nevertheless, different conditions were tested, and a counting of the colonies was done:
 +
 
 +
{| class="wikitable"
 +
|-
 +
!Condition
 +
!Gibson - usual mix - strain from Philippe
 +
!Gibson - usual mix - usual strain
 +
!Gibson - mix from Philippe - strain from Philippe
 +
!Gibson - mix from Philippe - usual strain
 +
!Plasmid alone - mix from Philippe - usual strain
 +
!Plasmid alone - mix from Philippe - strain from Philippe
 +
!Otherwise
 +
|-
 +
|Number of clones
 +
!2
 +
!25
 +
!0
 +
!$\infty$
 +
!3
 +
!2
 +
!0
 +
|-
 +
|}
 +
{{Team:Paris_Saclay/notebook_footer}}

Latest revision as of 17:04, 9 October 2016

Friday 9th September

Visualization

Colony PCR of 16 clones containing FRB - GFP 11 in pSB1C3 (pPS16_019)

By Maxence & Mahnaz

Colonies were obtained for the Gibson done the 7th September but not for the one done the 8th September. A colony PCR was done for 16 clones from the 7th September. For that purpose, 16 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, 4.13 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
61°C 30 sec
72°C 1 min 30 sec
Final Extension 72°C 7 min
Hold 4°C $\infinity\$

Primers used were:

Matrix Clones containing FRB - GFP 11 in pSB1C3 (pPS16_019)
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)
FRB - GFP 11 in pSB1C3 730
Result of the migration

All PCR products were at the good size and seemed to be concentrated. It could be explained as these clones were grown for 2 days. Clones 4, 9, 13 and 16 were selected and were grown at 37°C overnight.

PCR on GFP 1.9 in pUC19, gblock 1.1, gblock 1.2, gblock 2.1 and gblock 2.2 with 3% DMSO

By Maxence & Mahnaz

As the annealing temperature (Tm) seems too high to obtain good results for these amplifications, 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 amplification, two matrix were used: one was a previous PCR products and one was a plasmid.

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 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 PCR product of gblock 1.1 from 2nd September (Q5) gblock 1.1 9/08 PCR product of gblock 1.2 from 2nd September (Q5) gblock 1.2 11 PCR product of gblock 2.1 from 2nd September (Q5) gblock 2.1 PPS16003 PCR product of gblock 2.2 from 2nd September (Q5) gblock 2.2 PPS16004 PCR product of GFP 1.9 from 8th September GFP 1.9 in pUC19
Primers iPS140 and iPS120 iPS140 and iPS120 iPS121 and iPS122 iPS121 and iPS122 iPS123 and iPS124 iPS123 and iPS124 iPS125 and iPS84 iPS125 and iPS84 iPS84 and iPS140 iPS84 and iPS140

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)
gblock 1.1 960
gblock 1.2 960
gblock 2.1 1023
gblock 2.2 808
GFP 1.9 862
Result of the migration

All PCR products were at the good size.

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. For each amplification, only PCR products from plasmids were cleaned in order to reduce the risks of mutations. For GFP 1.9 amplification products, the 4 samples were pooled before clean up.

Results about FRB - GFP 11 transformed colonies from 8th September

By Maxence & Mahnaz

During the afternoon, observations were made about the 8th September Gibson, but as results were obtained for the 7th September Gibson, a colony PCR was not run. Nevertheless, different conditions were tested, and a counting of the colonies was done:

Condition Gibson - usual mix - strain from Philippe Gibson - usual mix - usual strain Gibson - mix from Philippe - strain from Philippe Gibson - mix from Philippe - usual strain Plasmid alone - mix from Philippe - usual strain Plasmid alone - mix from Philippe - strain from Philippe Otherwise
Number of clones 2 25 0 $\infty$ 3 2 0





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