Team:Stanford/Protocols
Lab Protocols
1: Reviving B. subtilis
Reviving Materials
- Plates
- Nutrient broth
- Sporulated Bacillus subtilis
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
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
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
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
Materials
- Water bath
- Plates
- LB media
- Competent E. coli cells
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
Materials
- 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
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
Materials
- 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
Materials
- Plates
- LB Agar
- Microwave
- Antibiotic
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
