Difference between revisions of "Team:Paris Saclay/Notebook/August/31"

(Created page with "{{Team:Paris_Saclay/notebook_header}} = Wednesday 31<sup>th</sup> August= ==Lab work== ===Visualization=== {{Team:Paris_Saclay/notebook_footer}}")
 
Line 3: Line 3:
 
==Lab work==
 
==Lab work==
 
===Visualization===
 
===Visualization===
 +
 +
====Colony PCR of 8 clones containing fragement 1 and 2 (dCas9 Nm GFP 10) in pSB1C3====
 +
''Mahnaz''
 +
 +
Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 8 clones containing dCas9 NM - GFP 10 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
 +
|8 min
 +
|-
 +
|rowspan="3"|30 cycles
 +
|95°C
 +
|30 sec
 +
|-
 +
|Tm
 +
|30 sec
 +
|-
 +
|72°C
 +
|t
 +
|-
 +
|Final Extension
 +
|72°C
 +
|10 min
 +
|-
 +
|Hold
 +
|4°C
 +
|infinity
 +
|}
 +
 +
[[Team:Paris_Saclay/Experiments#primers|Primers]] used were:
 +
 +
{| class="wikitable"
 +
|-
 +
!Matrix
 +
!Clones containing dCas9 NM - GFP 10 in pSB1C3
 +
|-
 +
|Primers
 +
|iPS83 and iPS84
 +
|-
 +
|Tm
 +
|60.2°C
 +
|-
 +
|t
 +
|4 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.
 +
 +
PCR products expected were :
 +
 +
{| class="wikitable"
 +
|-
 +
!PCR products
 +
!Expected band size (bp)
 +
|-
 +
|dCas9 NM - GFP 10 - pSB1C3
 +
|3688
 +
|}
 +
 +
  
  
 
{{Team:Paris_Saclay/notebook_footer}}
 
{{Team:Paris_Saclay/notebook_footer}}

Revision as of 13:30, 21 September 2016

Wednesday 31th August

Lab work

Visualization

Colony PCR of 8 clones containing fragement 1 and 2 (dCas9 Nm GFP 10) in pSB1C3

Mahnaz

Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 8 clones containing dCas9 NM - GFP 10 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 8 min
30 cycles 95°C 30 sec
Tm 30 sec
72°C t
Final Extension 72°C 10 min
Hold 4°C infinity 
Primers used were:
Matrix Clones containing dCas9 NM - GFP 10 in pSB1C3
Primers iPS83 and iPS84
Tm 60.2°C
t 4 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.

PCR products expected were :

PCR products Expected band size (bp)
dCas9 NM - GFP 10 - pSB1C3 3688







Université Paris Sud
91405 Orsay, France
Copyright © 2016 iGEM Paris Saclay. All rights reserved All inspirations encouraged.
*This wiki was conceived during long nights of work and a great coffee experience in the beautiful Orsay, France.