•   Home  
•   Team  
  • About Us
  • Profiles
•   Project  
  • Background
  • Future Implications
•   Data  
  • Protocols
  • Weekly Notebook
  • Bio Bricks
  • Results
•   Protocols  
  •  Molecular Cloning 
  •  pvdQ Expression Analysis 
•   Safety  
•   Attributions  
•   Human Practise  

pvdQ Expression Analysis Protocols

IPTG Induction

[PCR or Restriction Digestion Test must be performed to check for transformation of correct plasmid].

• Pick a colony and inoculate it into 5 mL broth with ampicillin. Incubate for 8 hours.

• Use 1mL of the culture in 10mL of broth with ampicillin (1:10 ratio). Grow the culture in warm room shaker till the OD reaches 0.6 (-0.8). Usually take around 3-6 hours.

• Add appropriate amount of IPTG (0.4-1.0 mM to the final concentration).

• Incubate at 37°C in shaker for 3-4 hours.

• Centrifuge and remove the supernatant (note the volume).

• Store the pellet in -20°C freezer until sonication.

 

Osmotic Shock to Obtain Periplasmic Fraction:

 

• Resuspend the pellet in 0.5mL PBS (phosphate buffered saline) or wash pellet twice in 30mM NaCl.
  [1L 1XPBS: 800mL Distilled Water, 8g NaCl, 0.2g KCl, 1.44g Na2HPO4-2H2O, 0.24g KH2PO4, adjust pH to 7.4 with HCl, Distilled water to a total of 1L. Isotonic and nontoxic to cells, so can be sued to dilute substances, wash reagent.]

• Centrifuge for 10min at 4°C (4863g). Discard the supernatant.

• Resuspend the pellet in 2.5mL ice-cold sucrose buffer.
  [Sucrose Buffer: 30mM Tris-HCl (pH 7.3), 1mM EDTA, and an equal volume of 0.5M sucrose.]

• Incubate for 10 minutes on ice while shaking vigorously.

• Centrifuge for 10min at 4°C (4863g). Discard the supernatant.

• Resuspend the pellet in 0.25 mL ice-cold 0.5mM MgCl2. Add appropriate amount of proteinase inhibitor.

• Incubate on ice for 10 minutes.

• Centrifuge for 20 minutes at 10,000g.

[Supernatant contains the periplasmic proteins. The pellet, which contains the rest of the cell components after the periplasmic components have been released into the supernatant, can be processed as the preparation of whole cell extracts. Then, the supernatant collected will contain the non-periplasmic cellular proteins.]