Difference between revisions of "Team:Paris Saclay/Notebook/September/19"

(PCR of pSB1C3 from dCas9 ST - GFP 11 clone 8 to correct mutations)
(Monday 19th September)
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===Visualization===
 
===Visualization===
  
==== Glycerol stocks of clones 7, 8, 9 and 10 containing FKBP - GFP 10 in pSB1C3 ====
+
==== Glycerol stocks of clones 7, 8, 9 and 10 containing FKBP - GFP 10 in pSB1C3 (pPS16_018) ====
 
"By Maxence, Mahnaz Coline & Caroline"
 
"By Maxence, Mahnaz Coline & Caroline"
  
 
The glycerol stock of the bacteria with the following plasmids were made.
 
The glycerol stock of the bacteria with the following plasmids were made.
*X (FKBP - GFP 10) clone 7
+
*pPS16_018 (FKBP - GFP 10) clone 7
*X (FKBP - GFP 10) clone 8
+
*pPS16_018 (FKBP - GFP 10) clone 8
*X (FKBP - GFP 10) clone 9
+
*pPS16_018 (FKBP - GFP 10) clone 9
*X (FKBP - GFP 10) clone 10
+
*pPS16_018 (FKBP - GFP 10) clone 10
  
 
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
 
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
  
====Plasmids extraction of clones 7, 8, 9 and 10 containing GFP 1.9 in pSB1C3====
+
====Plasmids extraction of clones 7, 8, 9 and 10 containing GFP 1.9 in pSB1C3 (pPS16_018)====
 
"By Maxence, Mahnaz Coline & Caroline"
 
"By Maxence, Mahnaz Coline & Caroline"
  
 
The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit:
 
The following plasmids were extracted using the [[Team:Paris_Saclay/Experiments#InvitrogenPlasmidExtraction|"Charge Switch-Pro Plasmid Miniprep"]] kit:
*X (FKBP - GFP 10) clone 7
+
*pPS16_018 (FKBP - GFP 10) clone 7
*X (FKBP - GFP 10) clone 8
+
*pPS16_018 (FKBP - GFP 10) clone 8
*X (FKBP - GFP 10) clone 9
+
*pPS16_018 (FKBP - GFP 10) clone 9
*X (FKBP - GFP 10) clone 10
+
*pPS16_018 (FKBP - GFP 10) clone 10
  
====PCR of pSB1C3 from dCas9 ST - GFP 11 clone 8 to correct mutations ====
+
====PCR of pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 to correct mutations ====
 
''By Maxence, Mahnaz, Coline & Caroline''
 
''By Maxence, Mahnaz, Coline & Caroline''
  

Revision as of 13:07, 19 September 2016

Monday 19th September

Lab work

Visualization

Glycerol stocks of clones 7, 8, 9 and 10 containing FKBP - GFP 10 in pSB1C3 (pPS16_018)

"By Maxence, Mahnaz Coline & Caroline"

The glycerol stock of the bacteria with the following plasmids were made.

  • pPS16_018 (FKBP - GFP 10) clone 7
  • pPS16_018 (FKBP - GFP 10) clone 8
  • pPS16_018 (FKBP - GFP 10) clone 9
  • pPS16_018 (FKBP - GFP 10) clone 10

500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.

Plasmids extraction of clones 7, 8, 9 and 10 containing GFP 1.9 in pSB1C3 (pPS16_018)

"By Maxence, Mahnaz Coline & Caroline"

The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:

  • pPS16_018 (FKBP - GFP 10) clone 7
  • pPS16_018 (FKBP - GFP 10) clone 8
  • pPS16_018 (FKBP - GFP 10) clone 9
  • pPS16_018 (FKBP - GFP 10) clone 10

PCR of pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 to correct mutations

By Maxence, Mahnaz, Coline & Caroline

In order to correct mutations in dCas9 ST - GFP 11, PCR was performed with the following protocol.

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 HF (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
72°C 30sec
72°C t
Final Extension 72°C 5min
Hold 4°C $\infty$
Primers used were:
Matrix dCas9 ST - GFP 11 clone 8 dCas9 ST - GFP 11 clone 8
Primers iPS174 and iPS175 iPS173 and iPS176
Tm 72°C 72°C
t 3min 50sec