Difference between revisions of "Team:Aix-Marseille/Notebook"

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==Training==
 
==Training==
  
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During the first week, we had several training sessions to get acquainted with the lab work and master everyday protocols (PCR/Colony-PCR, competent cells preparation, transformation, cloning, minipreps, clean-ups...).
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During the first week, we had several training sessions to get acquainted with the lab work, master everyday protocols (PCR/Colony-PCR, competent cells preparation, transformation, cloning, minipreps, clean-ups...), and establish a classic workflow.
  
 
====Preparing competent cells stock and transformation====
 
====Preparing competent cells stock and transformation====
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The next day we had colonies on our Petri dishes and wanted to check if they acquired the transformed plasmid with the right inserted gene. We picked out some positive colonies and ran a colony-PCR. We then migrated the PCR product on an agarose gel.
 
The next day we had colonies on our Petri dishes and wanted to check if they acquired the transformed plasmid with the right inserted gene. We picked out some positive colonies and ran a colony-PCR. We then migrated the PCR product on an agarose gel.
  
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====MINIPREP===
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====DNA purification miniprep===
  
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in the following daytime, we realized Miniprep to extract the DNA plasmidique. After this stage of purification of the DNA, we measured the quantity of DNA thanks to the nanobot . Five positive clones for the presence of insert are used according to the results of the colony PCR, and a negative clone (without band at 600) is used as negative control.
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We purified the plasmid with the right inserted gene from the positive using a DNA purification kit.
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And then, we have to realize the third check, by digesting the DNA plasmidique cleansed. An analysis on frost of electrophoresis is made, to verify the presence of the insert.
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In a further step, we digested the purified plasmid with respective restriction enzymes to double-check the presence of the inserted gene.
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OBTAINED RESULTS: a single clone has proved to be good, by the obtaining of the band at 600 bases after digestion. Other clones then are to be eliminated.
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The positive clone could be used for the sequencing for finally that we are absolutely sure that the band at 600 bases is good the insert.
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====Western-Blot====
 
====Western-Blot====
  
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the last day, we made a check of overproduction of a protein of a system inductible, by the analysis on frost of agarose in 17 %, under the classic method of purification of proteins, before induction, after induction and with the method of the benzonase with or without induction.
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The last day, we checked the induced production rate of the relative protein to the inserted gene (Produced in a preliminary step by our instructor) using the following western blot protocol.
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RESULTS OF THE WESTERN BLOT: on the Western Blot, we should obtain the band of the protein of interest only after induction, except us not induction of the production in case of the benzonase obtained the protein of interest with induction and, because there is always a flight of production of the protein even if she is not led by the molecular reactive of the promoter of the protein of interest. Furthermore, the use of the benzonase seems to increase the efficiency of migration in the frost. The benzonase degrades the DNA and seen that the protein of interest is a factor of transcription, her is bound to the DNA, and if the DNA is not degraded as in the case of the classic method, and good it prevents the good migration of the protein.
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Furthermore, the obtained bands are clearer, because the protein of interest better migrated. And it is also due to the stage of denaturation, the stage of TSTD in 95°C is more effective than 10 minutes in 37°C.
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BLUE: as regards the method of the Blue, we were able to observed the band of interest in the case only of the induction of the production of the protein. The benzonase seems increase the efficiency of the Blue to settle on protein for a better revelation of the studied protein of interest.
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Revision as of 13:03, 24 July 2018

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WEEK 1

Training

During the first week, we had several training sessions to get acquainted with the lab work, master everyday protocols (PCR/Colony-PCR, competent cells preparation, transformation, cloning, minipreps, clean-ups...), and establish a classic workflow.

Preparing competent cells stock and transformation

We prepared a stock of competent cells using the following protocol. Afterward, we prepared Petri dishes and we transformed the cells to test their efficiency using a plasmid provided by our instructor.

Colony-PCR and agarose gel electrophoresis

The next day we had colonies on our Petri dishes and wanted to check if they acquired the transformed plasmid with the right inserted gene. We picked out some positive colonies and ran a colony-PCR. We then migrated the PCR product on an agarose gel.

=DNA purification miniprep

We purified the plasmid with the right inserted gene from the positive using a DNA purification kit. In a further step, we digested the purified plasmid with respective restriction enzymes to double-check the presence of the inserted gene.

Western-Blot

The last day, we checked the induced production rate of the relative protein to the inserted gene (Produced in a preliminary step by our instructor) using the following western blot protocol.