Difference between revisions of "Team:Paris Saclay/Notebook/July/12"

(Created page with "=Monday 12<sup>th</sup> July= ==Lab work== ===Getting DNA closer=== ====Amplification of DS-TDcasN, DS-SPcasN and pZA21 plasmide==== ''By Caroline'' Plasmids extracted the 4...")
 
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=Monday 12<sup>th</sup> July=
 
=Monday 12<sup>th</sup> July=
 
==Lab work==
 
==Lab work==
  
 
===Getting DNA closer===
 
===Getting DNA closer===
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====Amplification of DS-TDcasN, DS-SPcasN and pZA21 plasmide====
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====Amplification of DS-TDcasN-, DS-SPcasN- and pZA21 plasmids====
 
''By Caroline''
 
''By Caroline''
  
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''By Charlene''
 
''By Charlene''
  
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Cells transformed the 11/07/16 have grown all the night. However no colony have been observed.  
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Cells transformed on 11/07/16 grew overnight. However no colony was observed.  
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To control if there is a bacteria transformed which have been not putted on Petri dish, we performed another experiment. 100 µl (500µl concentrated) of transformed bacteria for each condition, conserved at -4°C from yesterday, have been split in 3 solutions :
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To control if there is a bacteria transformed which was not put on Petri dish, we performed another experiment. 100 µL (500µL concentrated) of transformed bacteria for each condition, conserved at -4°C from yesterday, were split in 3 solutions :
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- 30µl of bacteria + 3ml LB + Streptomycin (50µg/ml) + Chloramphenicol (30µg/ml)
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* 30µL of bacteria + 3mL LB + Streptomycin (50µg/mL) + Chloramphenicol (30µg/mL)
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- 30µl of bacteria + 3ml LB + Streptomycin (50µg/ml)  
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* 30µL of bacteria + 3mL LB + Streptomycin (50µg/mL)  
−
- 30µl of bacteria + 3ml LB + Chloramphenicol (30µg/ml)
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* 30µL of bacteria + 3mL LB + Chloramphenicol (30µg/mL)
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They incubated 8h at 37°C, 200 RPM.  
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Cells were incubated for 8h at 37°C, 200 RPM.  
  
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No bacteria have grown so we determinated that it was a problem of transformation.
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No bacteria grew so we concluded that there was a problem with the transformation.
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{{Team:Paris_Saclay/notebook_footer}}

Revision as of 13:17, 12 July 2016

Monday 12th July

Lab work

Getting DNA closer

Amplification of DS-TDcasN-, DS-SPcasN- and pZA21 plasmids

By Caroline

Plasmids extracted the 4/07/16 were used to amplify the dCas9 needed for the get DNA closer tool and the pZA21 plasmid in which the tool will be inserted. A PCR was carry out following PCR with Q5® High-Fidelity 2X Master Mix from NEB protocol (cf protocols section) adapted to have 50µL at the end.

Biobrick characterization

Electrocompetents and transformation by electroporation of BL21

By Charlene

Cells transformed on 11/07/16 grew overnight. However no colony was observed. To control if there is a bacteria transformed which was not put on Petri dish, we performed another experiment. 100 µL (500µL concentrated) of transformed bacteria for each condition, conserved at -4°C from yesterday, were split in 3 solutions :

  • 30µL of bacteria + 3mL LB + Streptomycin (50µg/mL) + Chloramphenicol (30µg/mL)
  • 30µL of bacteria + 3mL LB + Streptomycin (50µg/mL)
  • 30µL of bacteria + 3mL LB + Chloramphenicol (30µg/mL)

Cells were incubated for 8h at 37°C, 200 RPM.

No bacteria grew so we concluded that there was a problem with the transformation.





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