Timp:ecoliProtocol

Home        Contact        Protocols        Lab Members        Publications        Research       

E.coli Treatment Protocols

Includes all protocols for culturing, handling and treatment of E.Coli.

Plate Preparation

• Materials:
  • LB-Agar Medium capsules (Bio 101 Systems)
  • Ampicillin Ready-made Solution (Sigma A5354-10ML)
  • Kanamyacin solution from Kanamyacin Sulfate- (Shelton Scientific IB02120)
  • DNAse, RNAse free Distilled Water (Gibco 5288)
  • Para film

• Procedure:
  • To make approximately 5 plates:

1. 200ml DI water
2. 8 capsules LB Agar
3. Autoclave and let cool
4. Add appropriate anti-biotic (200μL) (Kanamaycin for 111’s, 203’s, Ampicillin for M1’s and both for Oscillator cells)
5. Pour in Petri dishes, let it settle and Para film.
6. Parafilm, and store at 4C.
7. To streak plates, touch single colony with inoculating loop and streak in zig-zag pattern. Leave in warm room (37 C) over night.

Sample Preparation

• Materials:
  • 14ml Polystyrene Round-Bottom Tube (Falcon 352057)
  • Inoculating loops (Fisherbrand 14-375-103)
  • M9 Media       
• Procedure:

1. Touch single colony with inoculating loop and put it in 5mL of M9 media in round bottom tube.
2. Leave it in the warm room shaker over night.
3. Before the experiment dilute the saturated culture 1:5 and let it sit in the RT shaker for at LEAST 2hrs.
4. Spin it down at 1.4krpm for 2 min and re-suspend the pellet in M9 with appropriate antibiotic and let it sit in a vial for 15 min.
5. Spin it down again and gently re-suspend in M9 with no antibiotics.

Freezing E.coli in Glycerol Solution

• Materials:
  • Glycerol - Sigma G2025-500mL
  • Magnesium sulfate - Sigma M2643-500g
  • Tris*Cl pH 8
  • 2.0 ml Cyrogenic Vial (Corning-430659)
• Procedure:

1. Add ingredients in the following concentrations (brackets show values for 50mL):
   1. 60% Glycerol (30mL)
   2. 0.1M MgSO4 (0.602g)
   3. 0.025M Tris *Cl pH 8 (0.197g)
   4. Rest is 18 Mohm water (20mL)
2. Mix 1mL of freshly saturated culture with 1mL of glycerol solution in cyro Vial
   • (an overnight culture will do here)
3. Immediately store vial in -80C freezer

Thawing Glycerol Frozen E.Coli

• Materials:
  • LB Agar plates (refer to Plate Preparation protocol above)
  • Frozen Cell Culture
  • Inoculating Loop
  • Parafilm
• Procedure:

1. Scrape out a splinter or two of frozen culture and streak onto plate.
2. After Splinter has thawed, streak with inoculating loop
3. Grow at 37 C overnight. Plate should be inverted at ALL times.
4. Next day examine plate. Plates may be stored wrapped in Parafilm at 4C for less than one month safely.