Difference between revisions of "Team:Paris Saclay/Notebook/September/27"

(Created page with "{{Team:Paris_Saclay/notebook_header}} = Tuesday 27<sup>th</sup> September= ==Lab work== ===Visualization=== ====PCR of extracted GFP 1.9 in pSB1C3 (pPS16_020)==== ''By Maxen...")
 
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''By Maxence & Mahnaz''
 
''By Maxence & Mahnaz''
  
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In order to verify if extracted plasmids contain GFP 1.9, a PCR was run with iPS168 & iPS169 to amplify the insert.  
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In order to verify if extracted plasmids contain GFP 1.9, a PCR was run with iPS168 & iPS169 to amplify the insert. For that purpose, plasmids sent for sequencing the 14th September (clones 2, 7, 8 and 12) and plasmids extracted the 23rd Sepembter (clones 3, 4, 7 and 8) were used.
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For each 50μl of reaction, mix the following reagents :
 
For each 50μl of reaction, mix the following reagents :
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!Matrix
 
!Matrix
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!Extracted plasmid pSB1C3 containing GFP 1.9 (pPS16_020) from clones 3, 4, 7 & 8
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!Extracted plasmid pSB1C3 containing GFP 1.9 (pPS16_020)
 
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|Primers
 
|Primers

Revision as of 10:14, 27 September 2016

Tuesday 27th September

Lab work

Visualization

PCR of extracted GFP 1.9 in pSB1C3 (pPS16_020)

By Maxence & Mahnaz

In order to verify if extracted plasmids contain GFP 1.9, a PCR was run with iPS168 & iPS169 to amplify the insert. For that purpose, plasmids sent for sequencing the 14th September (clones 2, 7, 8 and 12) and plasmids extracted the 23rd Sepembter (clones 3, 4, 7 and 8) were used.

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
  • 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 95°C 30sec
30 cycles 95°C 30sec
64.4°C 30sec
72°C 30sec
Final Extension 72°C 5min
Hold 4°C $\infty$ 
Primers used were:
Matrix Extracted plasmid pSB1C3 containing GFP 1.9 (pPS16_020)
Primers iPS168 and iPS169

Gel of PCR products

By Maxence & Manhaz

4 µL of PCR products and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity.

GEL





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