Team:Pasteur Paris/Protocols/CellBio

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Molecular Biology: general protocols

Here we present the basic molecular biology methods we used throughout the project to amplify our plasmids, linearize them and insert our sequences, retrieve them from bacteria and express proteins.

Agarose Gel Preparation

Bacterial Stock

Gel Extraction

Ligation

Liquid Culture

Midiprep for plasmid extraction

Miniprep for plasmid extraction

Transformation of E. coli BL21 (DE3) pLysS

Transformation of E. coli DH5-alpha


Aim

Insert a plasmid of interest into competent bacterial cells, in order to replicate them.



Aim

Grow a colony that have successfully been transformed with one or several plasmids in order to replicate plasmid or to express a protein.



Aim

Stock bacterial culture at -80 °C.


Materials

  • Desired bacterial cultures on petri dish
  • Sterile LB media
  • chloramphenicol (25mg/mL) or carbenicilline(50mg/mL)
  • glycerol 50%

Procedure

In advance:

  • Prepare a stock solution of LB + desired antibiotic in 50 mL falcon tube depending on how many culture you would like to stock in glycerol.
  • Prepare a sterile stock solution of glycerol 50 %.
  1. In 15 ml sterile falcon, add 5 mL of LB media
  2. Vortex the stock solution of antibiotic and add 5 µL to the LB
  3. Using an inoculation loop, touch gently a colony of transformed bacteria from the petri dish, plastic side facing you.
  4. Immerse and dip the inoculation loop in the liquid media and stir.
  5. Place the liquid culture in the incubator at 37˚C 180 rpm for 16h.
  6. After 16 hours, centrifuge the tubes 5 minutes at 3000 rpm.
  7. Discard supernatant.
  8. Resuspend the pellet in 5mL of LB.
  9. Discard supernatant.
  10. Resuspend the pellet in 1 mL of LB + antibiotic.
  11. In a 125 ml erlenmeyer, add 1 mL of bacterial culture in 24 mL of LB + antibiotic.
  12. Incubate the culture at 37˚C 180 rpm.
  13. Measure the OD every hour for the first 3 hours and then every 20 minutes.
  14. When 0,6 < OD < 0,7, withdraw 5 mL of the bacterial liquid culture and add 5 mL of glycerol 50 %.
  15. Vortex.
  16. Aliquot the 10 mL into sterile cryotubes.
  17. Place into dry ice and freeze at -80˚C.

Get full protocol here



Aim

Retrieve amplified plasmids from a liquid culture of transformed bacteria. According to the liquid culture volume, we used the QIAfilter Plasmid Purification kit (for 25 mL culture) or the QIAprep Spin Miniprep kit (for 5 mL culture) from Qiagen.



Aim

Perform restriction enzyme digestion in order to recover linear backbones of the plasmids, extract our inserts from commercial plasmid, or check the success of a ligation.



Aim

Separate DNA fragments according to their molecular weight after an enzymatic digestion, in order to purify inserts or to analyse a plasmid.



Aim

: Extract DNA from an agar gel after an electrophoresis. We used the QIAquick Gel Extraction Kit provided by Qiagen.



Aim

Insert a DNA fragment with appropriate overlaps into a linearized plasmid. We used the In-Fusion HD Cloning Plus provided by Ozyme.