Team:UCL London/From the lab/Protocols

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Competent Bacteria

Materials:
  • 5×M9 salts in 500ml dH2O:
    • Na2HPO4--- 32g
    • KH2PO4 --- 7.5g
    • NaCl --- 1.25g
    • NH4Cl --- 2.5g
  • Minimal media: ( In 50ml Falcon )
    • Melted bacteriological agar solution (< 50°C) --- 39ml
    • 5×M9 salt solution --- 10ml
    • 20% (w/v) D-glucose --- 1ml
    • 1M CaCl2 --- 5µl
    • 1M MgSO4 --- 100µl
  • 0.1M CaCl2 / 15% glycerol: (In 59 ml Falcon)
    • 1M CaCl2 --- 5ml
    • 100% glycerol --- 7.5ml
Preparation:
Pour minimal media plates.
5x M9 salts.
Prepare 100ml LB per strain.
Prepare 50ml ice cold 0.1M CaCl2 / 15% glycerol per strain
Pre-chill eppendorf tubes
Method:
  1. Streak cells on minimal agar plate. Incubate 37°C overnight.
  2. Pick a colony into 5 ml LB + 100µl 1M MgSO4. Incubate 37°C overnight.
  3. Inoculate 100ml LB in pre-warmed conical with 1ml of the 5ml O/N culture from Step 2.
  4. Incubate 2 hrs in 37°C shaker until the cells at early log phase of growth curve (A600 ~ 0.3).
  5. Transfer to chilled, sterile two 50ml Falcon tubes and incubate on ice for 10 min.
  6. Cf 3300g 5 min in bechtop RmT. Cf.
  7. Resus in 10ml ice cold 0.1M CaCl2 / 15% glycerol and incubate on ice 30 min.
  8. Cf 3300g 5 min in benchtop RmT. Cf.
  9. Resus in 1ml ice cold 0.1M CaCl2 / 15% glycerol. Transfer 100µl aliquots to pre-chilled, pre-labelled eppendorf tubes. Store -80°C.


Transformation

Materials:
  • Plasmids DNA
  • Competent E.coli
  • NZY Medium
  • LB buffer
  • Ampicllin, Kanamycin, Tetracycline
  • Agar Plates
  • Eppendorf
Method 1:
  1. Add 5µL ice-cold plasmid DNA into the competent E.coli on ice.
  2. Incubate on ice for 30 min. (minimum) Incubate at 42°C for 45 sec.
  3. Incubate on ice for 2 min. (minimum)
  4. Add 300µL of NZYX medium.
  5. Incubate with shaking at 37°C for 1 hr.
  6. Spin the incubated E.coli suspension for 3min at maximum speed. (Optional)
  7. Discard about half of the liquid, and then re-suspend the E.coli solution. (Optional)
  8. Spread the suspension onto selective agar plates.
  9. Incubate at 37°C overnight.
  10. Pick a colony into 2ml selective LB (Add 100µL antibiotic to 50mL LB medium). Incubate 37°C in shaker overnight
Method 2:
  1. Using the puch tool, puch out the appropriate DNA. Clean the tool between punches.
  2. Soak the spots in 5µL of TE that has been warmed to 50ºC for 20 minutes. At this time take the competent cells from the -80º freezer and start defrosting on ice.
  3. Add 2µL of DNA in TE to 50µL of competent cells (TOP10)
  4. Allow the DNA and competent cells to sit on ice for 30 minutes
  5. Heat shock at 42ºC for 60 sec in water bath.
  6. Recover on ice for 5 min.
  7. Add 200 µL SOC media.
  8. Incubate at 37ºC for 2 hr while the tubes are rotating.
  9. Centrifuge and leave about 100 µL liquid; hence, resuspend the E.coli suspension.
  10. Plate 250µL on an LB plate with the appropriate antibiotic.
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