| NDSU - iGEM 2023
Protocol
  1. Thaw a tube of DH5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
  2. Add 2µl containing 1 pg-100 ng of Golden Gate reaction or plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  3. Place the mixture on ice for 30 minutes. Do not mix.
  4. Label and warm selection plates to 37°C.
  5. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  6. Place on ice for 5 minutes. Do not mix.
  7. Pipette 950 µl of room temperature SOC into the mixture.
  8. Shake at 37°C for 1-2 hours. Shake vigorously (250 rpm).
  9. Mix the cells thoroughly by flicking the tube and inverting, then perform two 10-fold serial dilutions in SOC.
  10. Spread 100µl of each dilution onto a selection plate and incubate overnight at 37°C.
Protocol
  1. Larger: 120ml 0.5x TAE buffer + 1.8g agarose, Smaller: 60mL 0.5x TAE buffer + 0.9g agarose. Microwave 40-80s, then 5 µL Midori Green
  2. Cast the gel in the gel rig if there is the rubber seal, or tape it if you can't find a rubber sealed one. Put in the comb.
  3. Ladder = 15µl “1Kb+ GeneRuler” Ladder + 3µl “Purple Loading Dye” dye.
  4. Samples (16x) = 25µl of Digest reaction + 5µl “Purple Loading Dye” dye. You can mix dye into the same tube we did the digest in.
  5. Once the gel is cooled, take out the comb and orient the gel so that the wells are on the black side. The DNA will “Run to Red”. Fill the gel rig with more 0.5x TAE until it fills and covers the wells.
  6. Add ladder + samples to wells.
  7. Run at 100V for 60 minutes.
  8. Take gel out and image with “UV365”.
Protocol
  1. Run the Golden Gate mixture in a thermocycler with the program listed in steps 2-4:
    Reagent Assembly Reaction (ng/Ul)
    DNA parts: Synthesized gene fragments; Plasmids designed for BsaI Golden Gate 40 fmol
    T4 DNA Ligase Buffer (10X) 2 µl
    NEB Golden Gate Enzyme Mix (BsaI-HFv2) 1.5 µl
    Nuclease-free H2O 20 µl
  2. 30 cycles of: 37°C for 1 minute and 16°C for 1 minute
  3. 60°C for 5 minutes
  4. Hold at 4°C
Protocol
  1. Add LB Lennox Broth powder to MiliQ water in a 20g powder / 1L water ratio. Only make as much as needed.
  2. Autoclave the mixture at Liquid20 setting.
  3. Once the media has cooled considerably (able to hold bottle comfortably), antibiotics can be added.
  4. The concentration of Amp (Ampicillin) in the media is always 100ug/mL unless noted otherwise.
  5. The concentration of Spc (spectinomycin) in the media is always 50ug/mL unless noted otherwise.
Protocol
  1. Make M9 salt solution by dissolving the appropriate amount of M9 salt into water. The ratio is listed on the bottle the M9 salts come in. Then autoclave the solution
  2. Make 20% glucose by dissolving 200g of glucose for each liter of water. Filter sterilize this mixture rather than autoclaving.
  3. Make a 1M stock solution for both MgSO4 and CaCl2. These two stocks can be autoclaved.
  4. For the biotin and thiamin, the ratio is 1mg/ml. To make a 50ml stock this would be 50mg. For the biotin small amounts of NaOH can be added to properly dissolve it. Both stocks should be filter sterilized rather than autoclaved, and stored at -20C
  5. Dissolve 5g of EDTA in 800ml of water. Next add 498mg of FeCl3, and 84mg of ZnCl2
  6. This next step requires stocks of CuCl2, CoCl2, H3BO3, and MnCl2 to have been premade, or can be added in mg amounts. MnCl2 is in very small milligram amounts so using stock solution would most likely be easier. Add 765uL of 0.1M CuCl2-2H2O, 210uL of 0.2M CoCl2-6H2O, 1.6mL of 0.1M H3BO3, and 8.1uL of 1M MnCl-4H2O.
  7. Solution is made, must be filter sterilized.
  8. For 5x M9 Salt solution add 200ml of the M9 salt solution.
  9. Next add 20ml of the glucose.
  10. Add 2ml of the 1M MgSO4 and 0.1ml of the 1M CaCl2.
  11. Add 1ml of the thiamine.
  12. Add 1ml of the biotin.
  13. Add 10ml of the trace element solution.
  14. Fill up the bottle to 1L using sterile water.
  15. This is the amount of reagent added to solution to create the specified final concentration
    Volume Reagent Final Concentration
    200 mL M9 Salt solution (5X) 1X
    20 mL 20% glucose 0.4%
    2 mL 1M MgSO4 2 mM
    0.1 mL 1M CaCl2 0.1 mM
    1 mL Biotin (1mg/ml) 1 ug
    1 mL Thiamin (1mg/ml) 1 ug
    10 mL Trace elements solution (100X) 1X
Protocol
  1. Add LB Lennox Broth powder to MiliQ water in a 20g powder / 1L water ratio. Only make as much as needed.
  2. Add agar to the the LB and water mix in a 15g agar / 1 L water ratio.
  3. Autoclave the mixture at Liquid20 setting.
  4. Once the media has cooled considerably (able to hold bottle comfortably), antibiotics can be added.
  5. The concentration of Amp (Ampicillin) in the media is always 100ug/mL unless noted otherwise.
  6. The concentration of Spc (spectinomycin) in the media is always 50ug/mL unless noted otherwise.
Protocol
  1. Pellet 4.5mL culture in a 1.5mL Eppendorf tube by spinning down 1.5mL at a time at 13000rpm and removing supernatant. Repeat 3-4 times. Or spin in a 15mL falcon tube for 10 minutes at 4000g.
  2. Resuspend cells in 250uL of buffer P1 (with RNAse added) in an Eppendorf tube
  3. Add 250uL Buffer P2 (if it is precipitate, warm the buffer in 30°C incubator) and mix by gently inverting 5 times, incubate at room temperature for exactly 5 minutes
  4. Add 350uL Buffer N3 and mix by inverting 5-10 times. Do not vortex. A white precipitant should begin to form.
  5. Spin for 10-15 minutes at 13000rpm in a centrifuge until good separation of precipitant and supernatant is achieved.
  6. Apply 800uL of clear supernatant to a QIAgen spin column. Avoid the white precipitate.
  7. Centrifuge for 60s, discard the flow through.
  8. Wash by adding 750uL Buffer PE (with ethanol added) and spinning for 60s. Remove flowthrough.
  9. Spin for an additional 2 minutes to remove residual PE buffer and discard the flowthrough.
  10. Transfer the top half of the spin column to a new Eppendorf tube. Add 50uL of ddH2O to center of the column, let sit for 3 minutes, then centrifuge for 60s. Discard the spin column.
  11. Label the Tube well with at least the following: Sample Name, Date, and Initials;
Protocol
  1. 10^-1 plate: 100 µL of original
  2. 10^-2 plate: 10 µL of original + 90 µL of S.O.C.
  3. 10^0 plate: Spin down remainder @ 13,000 rpm for 1 min, pour off supernatant, resuspend pellet in 100 µL of S.O.C., then plate all 100 µL
Protocol
  1. 2.5ul rCutSmart Buffer, 0.5µl EcoR, 0.5µl Cla1, 21.5µl DNA.
  2. Thermocycler: 37C for 40min then 65C for 20 min.
  3. 5 µL rCutSmart Buffer, 5µL Bsa1, 1 µg DNA (1000/concentration), water to 50 µL
  4. Thermocycler: 37°C for 5–15 minutes, 80°C for 40 min