Difference between revisions of "Team:Paris Saclay/Notebook/September/16"

(→‎PCR of pSB1C3 from dCas9 ST - GFP 11 clone 8 to correct mutations)
(→‎Colony PCR of 16 clones containing FRB - GFP 11 in pSB1C3)
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====Colony PCR of 16 clones containing FRB - GFP 11 in pSB1C3====
 
====Colony PCR of 16 clones containing FRB - GFP 11 in pSB1C3====
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''By Maxence & Mahnaz''
+
''By Mahnaz''
  
 
Colonies were obtained for the Gibson done the 15th September. A colony PCR was done for 16 clones from the 15th 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 15th September. A colony PCR was done for 16 clones from the 15th 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.

Revision as of 08:01, 16 September 2016

Friday 16th September

Lab work

Visualization

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

By Mahnaz

Colonies were obtained for the Gibson done the 15th September. A colony PCR was done for 16 clones from the 15th 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 FKBP - GFP 10 in pSB1C3
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 757

GEL