Team:WashU StLouis/Experiments-Protocols

Experiments/Protocols

Preparation of materials

Transformation

  • Preparaton of Competent E. Coli cells
  • Transformation of E. Coli cells
  • Transformation of Cyanobacteria (Synechocystis PCC 6803)
  • Preparation of LB Media

    1. Fill a container to about 70% of its volume with distilled water(assuming the container volume is the total volume of media intended to be made).
    2. Add 10 g/L of Tryptone.
    3. Add 10 g/L of NaCl.
    4. Add 5 g/L of Yeast Extract.
    5. Stir and fill up remaining volume with distilled water.

    Preparation of LB Agar

    1. Fill a container to about 60%-70% of its volume with distilled water(assuming the container volume is the total volume of media intended to be made).
    2. Add 10 g/L of Tryptone.
    3. Add 10 g/L of NaCl.
    4. Add 5 g/L of Yeast Extract.
    5. Add 20g/L of Agar.
    6. Stir and fill up remaining volume with distilled water.

    Preparation of a Glycerol Stock

    1. Add 750 μL 50% glycerol to cryotube
    2. Add 750 μL liquid culture to cryotube
    3. Store in -80°C freezer

    Preparation of LB + Antibiotic (i.e. Amp, Cm, Kan, etc.) Plates

    1. Heat previously prepared LB agar so that it has completely melted.
    2. Add enough antibiotic solution to the LB agar so that there is 1 microliter of Ampicillin solution per 1 mL of LB agar.
    3. Add about 20 mL of the LB agar + antibiotic mixture into each empty plate, and make sure that the entire plate is covered.
    4. Let the mixture cool and harden and then store the plates at 4 deg. Celsius.

    Preparation of Ampicillin (Amp) solution

    1. Measure 100 mg of solid Ampicillin per mL of antibiotic intended to be made.
    2. Dissolve the Ampicillin in dH20 and fill to the volume intended.
    3. Filter the Ampicillin solution through a syringe with an appropriate filtration tip.
    4. Aliquot the solution into microcentrifuge tubes and store at -20°C.

    Preparation of Chloramphenicol (Cm) solution

    1. Measure 30 mg of solid Chloramphenicol per mL of antibiotic intended to be made.
    2. Dissolve the Chloramphenicol in 100% EtOH and fill to the volume intended.
    3. Filter the Chloramphenicol solution through a syringe with an appropriate filtration tip.
    4. Aliquot the solution into microcentrifuge tubes and store at -20°C.

    Preparation of Kanamycin (Kan) solution

    1. Measure 50 mg of solid Kanamycin per mL of antibiotic intended to be made.
    2. Dissolve the Kanamycin in dH20 and fill to the volume intended.
    3. Filter the Kanamycin solution through a syringe with an appropriate filtration tip.
    4. Aliquot the solution into microcentrifuge tubes and store at -20°C.

    Preparation of Spectinomycin (Spec) solution

    1. Measure 60 mg of solid Spectinomycin per mL of antibiotic intended to be made.
    2. Dissolve the Spectinomycin in dH20 and fill to the volume intended.
    3. Filter the Spectinomycin solution through a syringe with an appropriate filtration tip.
    4. Aliquot the solution into microcentrifuge tubes and store at -20°C.

    Preparation of 1L of BG-11 media

    1. Add 10 mL of 100X BG11-FAC to a 1L jar.
    2. Add 1 mL of 6mg/mL Ferric Ammonium Citrate.
    3. Add 1 mL 189 mM Sodium Carbonate.
    4. Add 1 mL 175 mM Potassium Hydrogen Phosphate.
    5. Add 4.6 g of TES and 8.4 g of 100 mM Sodium Bicarbonate.
    6. Fill to 1L with dH20.

    Preparation of E. Coli Overnight Cultures

    1. Add 5mL of LB into a culture tube.
    2. Add 5 microliters of the appropriate antibiotic.
    3. Use a pipette tip or inoculation loop to pick a single colony off of the plate.
    4. Dip the loop or tip into the LB-antibiotic mix.
    5. Incubate at 37 deg. Celsius.

    Preparation of 2x Noble Agar

    1. Fill container with dH20.
    2. Mix in noble agar at 40.0g/L.
    3. Add remaining dH20 to desired volume.
    4. Autoclave to sterilize.

    Preparation of Competent E. Coli Cells

    1. Thaw 50 microliters of cells on ice for 5 minutes.
    2. Fill an overnight culture tube with 5 mL of LB media.
    3. Touch the tip of a pipet to the appropriate cells (either in a tube or on a plate) and place the cells into the culture tube.
    4. Grow the culture overnight for 16-18 hours at 37 deg. Celsius.
    5. During the next day, add 100 microliters of culture and 900 microliters of LB media to a cuvette.
    6. Measure the optical density (OD) of the sample using a spectrophotometer.
    7. Dilute the culture with LB media so that the OD of a sample is about 0.08.
    8. Incubate the culture at 37 deg. Celsius for about 75 minutes.
    9. Measure the OD of a sample by diluting by two and doubling the reading given.
    10. Incubate the culture at 37 deg. Celsius for 15-30 minutes (depending on the OD measured).
    11. Repeat the previous two steps until the OD lies between 0.4 and 0.6.
    12. Split the culture into 50 mL centrifuge tubes and place them on ice.
    13. Spin the tubes at 3000 RPM for 10 minutes at 4 deg. Celsius.
    14. Resuspend each pellet of competent cells in 5 mL of TSS buffer and place the tubes back on ice.
    15. Aliquot 100 microliters of the cell solution into separate microcentrifuge tubes while they are on ice.
    16. Place the microcentrifuge tubes of cells into the -80 deg. Celsius storage.

    Transformation of E. Coli cells

    1. Thaw 100 microliters of competent cells on ice.
    2. Add 5 microliters of DNA (plasmids) to the thawed competent cells.
    3. Incubate the cells and DNA on ice for 30 minutes.
    4. Place the tubes with the cells and DNA in a 42 deg. Celsius bath for 45-60 seconds.
    5. Leave cells on ice for about 2-5 minutes.
    6. Add 450 microliters of LB media to the cells.
    7. Pellet out the cell by centrifuging at 6000 RPM for 60 seconds.
    8. Remove 350 microliters of the supernatant and resuspend the cells.
    9. Pipet 100 microliters of the cells onto the appropriate plate and spread the cells throughout the plate using inoculation loops

    Transformation of Cyanobacteria (Synechocystis PCC 6803)

    1. Use log phase culture (~2-3 days), check O.D.730, should be between 0.2 & 0.8 (higher limit with glucose).
    2. Spin down appropriate amount of culture (5K, 5 min) & resuspend in 1X BG11+FAC to at least an O.D.730 = 2.5.
    3. Add 1-5 μg DNA (in up to 20 μl Qiagen buffer EB or water) to 200 μl cell suspension & incubate in sterile tube (2 ml cryovials work well) on its side in the light incubator for 6 h (resuspend by flicking at 3 h, or shake continuously).
    4. Add 2 ml 1X BG11+FAC media, transfer to 15 ml conical tube and perform outgrowth for 24 h, shaking vigorously in light incubator.
    5. Add appropriate antibiotic to BG11 plates for selection (see above, can use ½ concentration for initial selection, then increase for outgrowths) .
    6. Plate 500 μl culture on plate + antibiotics, and place in light incubator—initial growth takes 1-2 weeks, outgrowth streaks grow up in around 1 week or less.

DNA Manipulation Processes

DNA Separation/Purification Techniques

UV Irradiation Test

Restriction Digestion

  1. Add 1 to 15 microliters of DNA solution to a microcentrifuge tube.
    • The volume of DNA solution depends on its concentration.
  2. Add 5 microliters of digestion buffer (usually CutSmart).
  3. Add ddH20 so that the total volume in the microcentrifuge tube is 48 microliters.
  4. Add 1 microliter of each of the two enzymes and pipette up and down to mix.
  5. Incubate the tube at 37 deg. Celsius for 2 hours.

Ligation

  1. Using the measure concentration of DNA for the backbone and insert, the number of base pairs in each segment of DNA, and the desired molar ratio, calculate and add the appropriate masses and volumes of insert DNA and plasmid backbones into a microcentrifuge tube.
  2. Add 1 microliter of Ligase Buffer.
  3. Add ddH20 so that the total volume becomes 9 microliters.
  4. Add 1 microliter of DNA ligase.
  5. Incubate the ligation mixture at room temperature for 2 hours.
  6. After two hours, the DNA can be transformed into competent cells or it can be stored at -20°C.

Polymerase Chain Reaction (PCR)

  1. Add the following reagents together in a PCR tube (50μL, 40μL, 10μL totals)
  2. 50μL 40μL 10μL Reagent
    22 17.6 4.4 ddH20
    4 3.2 0.8 DMSO (100%)
    10 8 2 betain (5M)
    10 8 2 5x Phusion Buffer
    1 0.8 0.2 dNTPs (10mM)
    1 0.8 0.2 FWD primer (10mM)
    1 0.8 0.2 REV primer (10mM)
    0.5 0.4 0.1 DNA template
    0.5 0.4 0.1 Phusion DNA polymerase
  3. Put PCR tube in thermocycler and set to desired temperature and time
  4. Start the thermocycler and wait until the process is complete.
  5. Run PCR product on gel and the extract desired band for later use

PCR Gradient

  1. Use the 10 microliter proportions from PCR (see above) and multiply by 13 so that there are 130 microliters of PCR solution.
  2. Aliquot the solution into 12 PCR tubes of 10 microliters each.
  3. Choose the gradient protocol on the thermocycler.
  4. Set the median temperature (usually the temperature at which the primers should anneal at) and the temperature span.
  5. Start the thermocycler and wait until the process is complete.
  6. Run PCR on a gel and determine the well with the brightest and most well-defined ban. Then, determine which temperature the PCR was run at.

Golden Gate Assembly

  1. Add 0.4 μL Cutsmart Buffer, 0.4 μL DpnI, and plasmid backbone into a PCR tube.
  2. Incubate at 37°C for 1 hour.
  3. DNA purification for post-DpnI mix to retrieve plasmid backbone.
  4. Add 100 ng of plasmid backbone, equimolar amount of each assembly piece, 1.5 μL of restriction enzyme buffer, 1 μL of ligase buffer, 1 μL of Type IIs restriction enzyme, 1 μL of T4 ligase, and ddH2O to equal a total of 15 μL.
  5. Run in the thermocycler for 50 cycles (3 minutes at 37°C, 4 minutes at 16°C) and 1 cycle (5 minutes at 50°C, 20 minutes at 80°C), and hold at 4°C.
  6. Transform the ligated DNA into competent cells.

Miniprep

  1. Incubate a cell culture overnight at 37 deg. Celsius.
  2. Transfer the culture to microcentrifuge tubes.
  3. Centrifuge the microcentrifuge tubes at 3000 RPM for 2 minutes and decant the supernatant.
  4. Resuspend the cell pellets in 250 microliters of resuspension solution.
  5. Add 250 microliters of lysis buffer.
  6. Add 350 microliters of neutralization solution and mix immediately.
  7. Centrifuge the tubes at 10,000 RPM for 15 minutes.
  8. Transfer to spin columns with microcentrifuges attached.
  9. Centrifuge for 1 minute at 10,000 RPM and discard the flow-through.
  10. Add 500 microliters of wash solution into the spin column.
  11. Centrifuge for 30-60 seconds at 10,000 RPM and discard flow-through
  12. Repeat the previous two steps.
  13. Centrifuge for 1 minute at 10,000 RPM.
  14. Transfer the spin columns onto new 1.5-mL microcentrifuge tubes and add 50 microliters of elution buffer.
  15. Incubate for 2 minutes at room temperature.
  16. Centrifuge for 2 minutes at 10,000 RPM.
  17. Discard the spin column and store the DNA solution in the microcentrifuge tube at -20 deg. Celsius.

Gel Electrophoresis

  1. Mix 50 mL of TAE (Tris-acetate-EDTA) buffer with a certain mass of agarose so that the mass concentration is 1-3%
    • The mass concentration depends on the size of the DNA fragments being separated in the electrophoresis.
  2. Microwave the solution so that the agarose dissolves in the TAE buffer.
  3. Pour the solution into the gel cassette.
  4. Add 5 microliters of Ethidium Bromide and mix it into the TAE-agarose solution.
  5. Place a comb onto the the cassette (to make the wells).
  6. Let the gel solidify for 30 minutes.
  7. Add 6x loading dye to the digestion mixture so that it makes up 1/6 of the final total volume.
  8. Place the gel with the cassette into the electrophoresis box and add TAE buffer so that it covers the entire gel.
  9. Remove the comb to expose the empty wells.
  10. Add the DNA ladder to the first well.
  11. Pipette the digestion mixture into the wells.
  12. Set the voltage to 120V and run the gel until the bands are distinct (about 30-45 minutes).
  13. Using the proper protection, view the bands with UV light, and cut out the wanted bands which can be stored at -20 deg. Celsius in a microcentrifuge tube.

Gel Purification

  1. Determine the mass of each gel fragment by weighing it and subtracting the mass of an empty microcentrifuge tube.
  2. Add BNL Buffer so that there is 300 microliters of buffer for every 100 mg of agarose gel.
  3. Heat and shake at 55°C for 10 minutes.
  4. Add the mixture to the spin column and collection tube, and centrifuge it at 13,000 RPM for 1 minute. Discard the flow-through.
  5. Add 700 microliters of Washing Buffer to the column and centrifuge at 13,000 RPM for 1 minute. Discard the flow-through.
  6. Centrifuge the column and tube once again at 13,000 RPM for 1 minute to dry the spin column.
  7. Replace the collection tube with a clean microcentrifuge tube, and add 20 microliters of Elution Buffer. Then, centrifuge the column and microcentrifuge tube at 13,000 RPM for 1 minute.
  8. Discard the spin column, and close the microcentrifuge tube. The eluted DNA can be stored at -20°C.

UV Irradiation Test in One Hour Intervals

  1. Grow 3 3-5ml overnight cultures in test tubes.
  2. Measure OD of all three and dilute to OD of 0.1 into culture tubes filled with 3 mL of LB.
  3. Grow on shaker, closely monitoring OD starting around 1.5 hours after starting the culture, and once one tube reaches an OD of 0.4 to 0.8, remove the tubes from the shaker, and add IPTG to each of the tubes.
  4. Grow induced cells for 3-4 hours and then remove from the shaker.
  5. For each test tube, fill a 1.5 mL microcentrifuge tube with 1 mL of cell solution.
  6. Spin tubes for 2 minutes at 8000 RPM to pellet the cells and remove the supernatant.
  7. Fill the tubes with 1 mL of 0.9% NaCl solution and resuspend cells.
  8. Repeat steps 6 and 7.
  9. Measure the OD of the cell solution in tubes using 900 microliters of LB and 100 microliters of cell solution.
  10. Fill 30 autoclaved 1.5 microcentrifuge tubes with 900 microliters each of 0.9% NaCl solution. Label these each of tubes as one of the three cell lines being tested, one of the five time points labeled (0, 1, 2, 3 or 4 hrs), and one of the two concentrations (10^(-1) or 10^(-2)) for each hour.
  11. Dilute the cells in glass petri dishes to a final concentration of 2x10^7 cells/mL with 6 mL of 0.9% NaCl solution.
  12. Move 100 microliters of solution from the plates to the tubes labeled "hour 0, dilution 10^-1" for the appropriate cell line and pipette to mix well.
  13. Move 100 microliters of solution in the tubes from step 12 into the corresponding tubes labelled "dilution 10^-2" and mix well.
  14. Move the plates to the environmental simulation system and line them up under the UV lamp, close the door, and turn the light on as the timer is set for one hour.
  15. While the cells are being exposed to UV, plate 10 microliters of the 10^-2 dilution cell solution for each cell line three times for a total of nine plates and leave them outside of the 37 degree room until the last time point.
  16. Repeat steps 12-15, for each of the time points using the appropriately labelled tubes.
  17. Once all of the samples from each time point have been plated, move all plates to the 37 degree room and grow overnight.
  18. During the following morning, count the number on each plate twice and take the average of the two as the cell count.

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