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

(→‎WEEK 1)
Line 4: Line 4:
 
==Training==
 
==Training==
  
−
During the first week, we had several training sessions to get acquainted with the lab work and master everyday protocols (PCR, competent cells preparation, transformation, cloning, minipreps, clean-ups...).
+
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...).
  
−
====Preparing competent cells stock====
+
====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.
 
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:
+
====Colony-PCR and agarose gel electrophoresis====
−
A PCR colony is realized to test the presence of the insert in the plasmid acquired by the bacterium. We selected controls colonies, colonies transformed with the digested DNA and colonies transformed with the plasmidic not digested DNA (circular). Once realized, we realized a migration of the DNA on gel of electrophoresis, for the analysis of the product of PCR.
+
 
−
EXPECTED RESULTS: size expected from the insert is 600 bases.
+
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.
−
In this case, we used the SacB system of identification of clones containing the insert in the plasmid: if SacB has amplified thanks to the primer 1 then the insert is not present in the plasmid. SacB is a very long DNA sequence, it will be then at the beginning of frost.  
+
 
−
If SacB is not then amplified,it is because there is an insert in the plasmid, and more particularly in the middle of the gene SacB. The insert will be then amplified by the primer 2.
+
====MINIPREP===
−
OBTAINED RESULTS: in every case, we obtained the plasmid and the primers. In the case of the controls, we were able to amplify SacB which is the highest band on the frost by its big size (see the well number 7 of Emeline). For other samples, we obtained a band in 600 bases which seems to correspond to our insert. In the case of certain samples, we sometimes obtain that the plasmid and the oligos what demonstrates the absence of insert in the plasmid of the clone.
+
−
NOTICE: it would have been necessary to realize a negative control with a well of migration without the matrix of DNA (without bacteria), it is the only condition of non-obtaining of bands.
+
−
A positive control could also be in realized with a clone containing the cleansed plasmide.
+
  
−
==MINIPREP:==
 
 
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.
 
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.
 
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.  
 
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.  
Line 25: Line 21:
 
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.
 
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.
  
−
==CHECK OF THE PRODUCTION OF A PROTEIN INDUCIBLE STEMMING FROM A PLASMIDE:==
+
====Western-Blot====
  
 
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.  
 
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.  

Revision as of 12:51, 24 July 2018

HOME
TEAM
PROJECT
Description
Design
Experiments
Notebook
Protocols
InterLab
Model
Results
Perspectives
Safety
PARTS
Parts Overview
Basic Parts
Composite Parts
HUMAN PRACTICES
Integrated HP
Education & Engagement
Collaborations
ACKNOWLEDGEMENTS
Sponsors
Attributions
Crowdfunding
JUDGING FORM ⇗

{{{title}}}

WEEK 1

Training

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...).

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.

=MINIPREP

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. 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. 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. 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.

Western-Blot

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. 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. 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. 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.