Difference between revisions of "Team:Paris Saclay/Notebook/September/21"

(→‎Colony PCR of 16 clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022))
(→‎Wednesday 21st September)
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==Lab work==
 
==Lab work==
 
===Visualization===
 
===Visualization===
 +
 +
====Colony PCR of 16 clones containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)====
 +
''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.
 +
 +
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°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"
 +
|-
 +
!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 :
 +
 +
{| class="wikitable"
 +
|-
 +
!PCR products
 +
!Expected band size (bp)
 +
|-
 +
|dCas9 ST - GFP 11
 +
|3800
 +
|}
 +
 +
GEL
  
 
====Colony PCR of 16 clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)====
 
====Colony PCR of 16 clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)====
Line 67: Line 135:
 
!Expected band size (bp)
 
!Expected band size (bp)
 
|-
 
|-
−
|FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
+
|FRB - GFP 11 - GFP 1.9
 
|1500
 
|1500
 
|}
 
|}
 +
 +
GEL
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Revision as of 16:26, 20 September 2016

Wednesday 21st September

Lab work

Visualization

Colony PCR of 16 clones containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)

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 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 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

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

GEL

Colony PCR of 16 clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)

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 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 1500

GEL





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