Help:3A Assembly Kit/Growing

Introduction


Step #1: Growing

Your kit includes agar stabs for Part A (BBa_J04500) and Part B (BBa_J04650). The agar stabs contain live bacteria (E. coli) that house your parts. The parts themselves belong to a plasmid which replicates within the E. coli cell.

Before working with your parts, you'll need to make sure you have enough copies of them. You can do this by growing up cell cultures from the agar stabs: more E.coli cells means more plasmids, which means more copies of your parts. Use the provided agar stabs to streak out the bacteria containing your parts onto agar plates. Then pick a single colony to grow up a cell culture.

Part A and Part B are both maintained on pSB1AK3 plasmid backbones. The ‘AK’ means that your plasmid backbone is resistant to Ampicillin and Kanamycin antibiotics. When you’re growing these parts up, make sure to use media that has these antibiotics, that way you can ensure that only the bacteria containing your parts will grow.

The 3A Assembly Kit also includes an agar stab with the NEB 10-beta strain that you'll need to make competent cells. Click here for the Competent Cell Production Protocol and note that you'll need to begin the production protocol four days in advance to Step #5: Transformation. You can also use purchased competent cells for the Transformation step as well.


Streaking from agar stabs

estimated time: 15 min. active, 16-24 hrs. incubation

Materials needed

  • 70% ethanol
  • Paper towels
  • Lab marker/Sharpie
  • Agar Stab: Part A - BBa_J04500 (kit)
  • Agar Stab: Part B - BBa_J04650 (kit)
  • Inoculating loops (kit)
  • LB agar plates – Amp/Kan (kit)

Protocol

  1. Clean the lab bench by wiping down with 70% ethanol and paper towels.
  2. Part A (BBa_J04500) and Part B (BBa_J04650) are both maintained on pSB1AK3 plasmid backbones, which means they are ampicillin- and kanamycin-resistant. Label the agar plates with the names of Part A and Part B.
  3. Notice how each zig-zag overlaps with the previous one just a little, and only at the end.
    Use an inoculating loop to transfer some cells from the Part A agar stab to the appropriately labeled Amp/Kan agar plate. There is a hole in each agar stab from where it was inoculated. Dip an inoculating loop into the stab at the same location, and streak the bacteria onto the agar plate in a zig-zag pattern. Using a fresh inoculating loop, streak onto the agar plate again creating a new zig-zag pattern that overlaps the first. This will help ensure that you will have single colonies to pick from. Streak gently, and try not to puncture the agar.
  4. Repeat step 4 for Part B..
  5. This prevents other bacteria from settling, and growing, on your agar plate.
    Place the agar plates into the incubator with the agar side facing up, lid facing down (see insert). Incubate the agar plates at 37°C for 14-16 hours. Alternately, incubate at room temperature for 24-30 hours.
  6. Once your agar plates have grown up you can store them in your fridge (4°C) until you're ready to grow up your cell culture.
    1. Plates can be stored at 4°C for up to 3 weeks.


Growing up cell cultures

estimated time: 30 min. active, 16 hrs. incubation

Materials needed

  • 70% ethanol
  • paper towels
  • Lab marker/Sharpie
  • 14ml culture tubes (kit)
  • 10ml of LB broth - Amp/Kan (kit)
  • Inoculating loops (kit)
  • Agar plate: Part A – BBa_J04500 (see previous step)
  • Agar plate: Part B – BBa_J04650 (see previous step)
  • Rotator/Shaker

Protocol

  1. Clean the lab bench by wiping down with 70% ethanol and paper towels.
  2. Remove the agar plates for Part A and Part B from the incubator or 4°C fridge.
  3. Label one 14ml culture tube for each Part. Add 5ml of LB broth (with ampicillin and kanamycin) to each culture tube.
  4. Use an inoculating loop to pick a single colony from each agar plate and inoculate the LB broth, in the appropriately labeled culture tube. Do not use the same inoculating loop more than once! Press lightly on the snap caps of the 14ml tubes, the caps should be a bit loose to allow for air flow.
  5. Incubate for 16 hours at 37°C, in a rotator or shaker. Rotation helps the cells grow faster, and prevents them from settling at the bottom.
  6. After incubation, the cell culture should be cloudy. You can now firmly press down on the snap caps to seal the tubes and store the cell culture at 4°C until you're ready to move onto the next step.


Preparing NEB 10-beta cells

For instructions on how to prepare NEB 10-beta cells (i.e. competent cells), see the Competent Cell Production Protocol. You will need these cells for Step #5: Transformation. The production protocol requires several days, so plan accordingly!


Step #2: Miniprep