Protocols Engineering

Lab Protocols

1: Reviving B. subtilis

Reviving Materials

  • Plates
  • Nutrient broth
  • Sporulated Bacillus subtilis

Reviving Spores:

Total time: 12+ hours

  • Add spores to selective media
  • Add several drops of nutrient broth to cover spores
  • Grow for a minimum of 12 hours at 37°C

2: Extracting Plasmid DNA (MiniPrep)

MiniPrep Materials

  • 2mL tubes
  • Qiagen Miniprep Kit
  • Centrifuge


Total time: 35 minutes

  • Spin down overnight E. coli culture at 3500 rpm for 8 minutes
  • Aspirate media from cell pellet
  • Resuspend pellet in 250 μl Buffer P1 and transfer to 2mL tube
  • Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Do not allow this step to proceed for more than 5 minutes
  • Add 350 μl Buffer N3. Mix immediately and thoroughly by inverting the tube 4–6 times
  • Centrifuge for 10 min at 13,000 rpm
  • Add 800 μl of the supernatant from step 6 to the QIAprep 2.0 spin column
  • Centrifuge for 30–60s, discard the flowthrough
  • Add 750 μl Buffer PE to spin column and centrifuge for 30–60s
  • Discard the flow-through, and centrifuge at full speed for an additional 1 min
  • Label the side of new 2mL tubes, then cut the tops off the tubes with scissors. This prevents tops snapping off in the centrifuge
  • Place the QIAprep 2.0 column in the topless 2mL tube. Add 50 μl Buffer EB to the center of each QIAprep 2.0 spin column, let stand for 1 min and centrifuge for 1 min at 13,000 rpm
  • Cap the tube and label. Nanodrop to find DNA concentration, using Buffer EB as a blank

3: PCR

PCR Materials

  • Thermocycler
  • GoTaq PCR MasterMix
  • Nuclease free water
  • Primers
  • DNA


Total time: 4-8 hours

  • In PCR tubes, add the following: 10ul GoTaq, 1uL each of primers, 2ul DNA, then bring to 20ul with nuclease free water
  • Put in thermocycler with the following settings: Initial denaturation, 98°C, 1min, 1x. Denaturation, 98°C, 15s, 25-30x. Annealing, Tm of primers - 2°C, 30s, 25-30. Extension, 72°C, 1 min/1000bp, 25-30x. Final extension, 72°C, 7min, 1x.

4: Transformation in B. subtilis

Competence Materials

  • Competent B. subtilis cells
  • Plasmid
  • LB medium
  • LB agar plate with selective media
  • Spectrophotometer
  • Xylose or mannitol
  • Incubator at 37°C

1A967 Transformation:

Total time: 4.5 hours

  • Dilute overnight culture into 5mL fresh media at 1.0 OD with 10mg Xylose per mL or inoculate cells from plate into media to 1.0 OD with 10mg Xylose per mL
  • Grow for 3 hours at 37°C
  • Add DNA, incubate for 1 hour at 37°C. For best results, add at least 100ng DNA/mL
  • Plate on selective media

1A1267 Transformation:

Total time: 4.5 hours

  • Dilute overnight culture or inoculate cells from plate into media to 0.1 OD
  • Grow for 1.5 hours at 37°C
  • Add 5mg Mannitol per mL to the liquid culture
  • Grow for 1.5 hours at 37°C
  • Pellet cells at 3500rpm for 8 minutes
  • Aspirate media
  • Resuspend in fresh media to 0.5 OD
  • Add DNA
  • Incubate for 1 hour at 37°C
  • Plate on selective media

5: Gibson Assembly

Gibson Materials

  • Gibson Assembly Mastermix
  • Backbone
  • Inserts
  • DI Water
  • 50°C heating block

Gibson Assembly:

Total time: 25-75 minutes

  • For a 2-3 fragment assembly, mix 0.2 pmols of fragments and 10 uL of Gibson Assembly Master Mix. Bring to 20 uL with DI water
  • Incubate at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled
  • Store at -20°C

6: Transformation Into E. coli


  • Water bath
  • Plates
  • LB media
  • Competent E. coli cells

Tranformation Into E. Coli:

Total time: 2 hours + overnight

  • Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).
  • Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 20-50 μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.
  • Incubate the competent cell/DNA mixture on ice for 20-30 mins.
  • Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 secs (45 secs is usually ideal, but this varies depending on the competent cells you are using).
  • Put the tubes back on ice for 2 min.
  • Add 250-1,000 μl LB or SOC media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min.
  • Plate some or all of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic.
  • Centrifuge for 30–60s, discard the flowthrough
  • Incubate plates at 37°C overnight.

7: Running Agar Gels


  • Agarose
  • SybrSafe DNA Stain
  • LB media
  • DNA
  • DNA ladder
  • Loading dye (if not using green GoTaq PCR MasterMix)
  • Gel electrophoresis box
  • Microwave
  • UV gel visualizer
  • 1x TAE buffer

Gel Electrophoresis:

Total time: 1-2 hours

  • To prepare the gel, add 0.5-2g agarose to 100mL 1x TAE Buffer. Use less agarose for larger fragments
  • Microwave in 45 second intervals until agarose is fully dissolved
  • Add 5uL SybrSafe DNA Stain
  • Pour gel and add well comb
  • Wait 15+ minutes for gel to solidify
  • Remove comb
  • Place gel in electrophoresis box and fill box with 1x TAE until gel is submerged
  • Pipette 10-20uL of sample into each well. Add ladder to one well
  • Run gel at 100-180 mV until dye is ¾ through gel (20-40 minutes)
  • Visualize using UV gel visualizer

8: Sporulating B. subtilis


  • 3+ day old culture of Bacillus subtilis
  • Heating block
  • Liquid media


Total time: 20 minutes (after cultures grow)

  • Allow Bacillus subtilis cultures to grow for 3-5 days (or until they reach their decline phase of growth) at 37 degrees Celsius°C and 200 rpm
  • Prepare serial dilutions of the culture (10-2 - 10-6 fold) with liquid media
  • Incubate at 80°C for 15 minutes
  • Store spores at room temperature in tubes or aluminum foil

9: Making Agar Plates & Nutrient Broth


  • Plates
  • LB Agar
  • Microwave
  • Antibiotic

Pouring Plates:

Total time: 30 minutes

  • Warm agar in microwave until agar has dissolved
  • Wait for the agar to cool until can be touched without burning
  • Add the antibiotic
  • Pour plates about halfway up the plate wall
  • Let cool 15+ minutes
  • Store plates upside down at 4°C

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