IGEM:VGEM/2007/Notebook/2007-6-6

• Biobrick Construction Tutorial
  • Transformation of E. coli with BBa_I13522 from Plate 3, Well 13C.
  • Cell type: subcloning efficiency DH5alpha
    
• What was done:
  • Bacteria were transformed to express GFP (with the aforementioned Biobrick)
  • 20 uL and 200 uL of transformed bacteria were plated and incubated overnight.
  • 20 uL and 200 uL of nontransformed bacteria were plated and incubated overnight as a control.
  • Total of 4 plates
  • ~5 uL of suspended Biobrick leftover

Protocol:

Protocol for Transforming E. Coli with Provided Biobrick

Updated: June 7, 2007

Preparing LB broth and agar:

  1. Add 10g of Tryptone, 10g of NaCl, and 5g of yeast per liter of diH2O
  2. Adjust pH to 7.4 (usually by adding NaOH)
  3. Set aside 100 mL of LB broth for overnight cultures
  4. Add 18g agar per 1 liter
  5. Autoclave

Preparing Ampicillin:

  1. Add 100mg ampicillin powder per 1ml water
  2. Filter mixture through nitrocellulose membrane
  3. Add 1mL of ampicillin mixture to 1L of agar mixture

Pouring plates:

  1. After autoclaved agar mixture cools, pour directly into Petri dishes
  2. Wait 2-3 hours for plates to set, store upside down and labeled

Getting the DNA out of the Biobrick wells:

  1. Puncture a hole through the foil with a pipette tip into the well that corresponds to the Biobrick™-standard part that you want (We used BBa_I13522 for this, a reporter part which expresses GFP and provides ampicillin resistance)
  2. Add 15 uL of diH2O (deionized water) to the well

Transforming Cells:

  1. Thaw ~50 μl cells on ice. Do not use glass tubes, which adsorb DNA.
        1. Thaw another 50 μl of cells for the control plates (these will not be transformed)
  2. Add 1 ul DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
  3. Incubate all cells on ice for 30 minutes.
        1. Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
  4. Incubate cells for 50 seconds at 42°C.
  5. Incubate cells on ice for 2 min.
  6. Add 1mL of LB broth to a tube and add all of the cells into it
  7. Incubate for 1 hour at 37°C on shaker. 
        1. Note: Can also save some time here by reducing incubation to ~45 min.
        2. Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
  8. Sterilize the spreader
  9. Spread 20 & 200 μl of transformed cells and control cells onto plates made with appropriate antibiotic (i.e. 100mg/ml of Amp), four plates in total.
 10. Grow overnight at 37°C.

Making LB broth mixture for miniprep and glycerol:

  1. Pick a single colony with inoculating loop
  2. Add to 5mL of LB broth (containing 5μg/ml Amp) and grow  ~18 hours @ 37C
  3. Use the resulting culture to miniprep the DNA and make your own glycerol stock

Making glycerol stock for long term storage:

  1. Add 1 ml of 40% glycerol in H2O to a cryogenic vial.
  2. Add 1 ml sample from the culture of bacteria to be stored.
  3. Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.
        1. Alternatively, pipet to mix.
  4. On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
  5. Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later.

ERuhi 21:37, 8 June 2007 (EDT)