Team:TUDelft/ExperimentsTest

Sci-Phi 29

  • General ﹀
    • Antibiotic Stock Solution ﹀
      1. Weigh X gram of the antibiotic of interest.
      2. Dissolve the antibiotic of interest in Milli-Q water. The volume of Milli-Q water needed is determined by the desired antibiotic concentration.

      Note: Dissolve in 95% ethanol in case of chloramphenicol.

    • DNA Gel Electrophoresis ﹀

      The protocol for DNA gel electrophoresis includes mutagenic chemicals such as ethidium bromide (EtBR), SYBR Safe or another DNA staining. Carry out the protocol in an assigned area and wear gloves.



      Gel preparation:
      1. Prepare 0.8% (weight/volume) agarose in Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE) buffer.
      2. Heat the gel solution in the microwave to dissolve the agarose. Swirl the solution a few times while heating to ensure that the agarose powder is dissolved. The agarose is dissolved when the solution has bubbles and is clear.

      Casting of gel:
      1. Pick an appropriate gel stand and gel comb and place in the gel casting stand. Make sure to fasten the stand in place. Perform the next few steps quickly to prevent the gel from solidifying.
      2. Spread 15 µL (for a large stand) or 4-5 µl (for a small stand) of SYBR Safe or EtBr on the stand.
      3. Carefully pour the gel into the stand. Ensure that 3/4 of the teeth of the comb are submerged in the solution.
      4. Allow the gel to solidify. This will take approximately 30 minutes.

      Sample loading:
      1. After the gel has solidified, carefully remove the comb and transfer the gel to a gel box filled with TAE or TBE buffer.
      2. Cut a strip of parafilm. Pipette a drop of loading dye onto the parafilm, then pipette the sample on top of the loading dye. The ratio should be 1:5::dye:sample (e.g. 1 µL of dye and 5 µL of sample). Mix the solutions by pipetting up and down a few times. Repeat this step for each sample.
      3. Load the samples carefully in the slots. Make sure not to poke the gel.
      4. Load 3 µL of DNA ladder in a separate slot.
      5. Set the voltage to 100 volt.
      6. Run the gel until the DNA has moved across 2/3 of the gel. This will take approximately 45 minutes.
      7. Visualize the gel using a gel documentation system (gel doc).
    • DNA Gel Purification ﹀

      Protocol for PCR clean-up using Promega Wizard® SV Gel and PCR Clean-Up System:



      Dissolving the gel slice:

      1. Following electrophoresis, excise a DNA band from the gel and place this gel slice in a 1.5 mL microcentrifuge tube.
      2. Add 10 µL of Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50–65°C until the gel slice is completely dissolved.

      Binding of DNA:

      1. Insert a Wizard® SV Minicolumn into a Collection Tube.
      2. Transfer the dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
      3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert the Minicolumn into the Collection Tube.

      Washing:

      1. Add 700 µL of Membrane Wash Solution. Membrane Wash Solution should be prepared beforehand with 15 mL of 95% ethanol added per 3 mL of concentrated Membrane Wash Solution. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert the Minicolumn into the Collection Tube.
      2. Repeat step 1 with 500 µL of Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.
      3. Empty the Collection Tube and centrifuge the column assembly again for 1 minute at 16,000 x g with the microcentrifuge lid open or off to allow evaporation of any residual ethanol.

      Elution:

      1. Carefully transfer the Minicolumn to a clean 1.5 mL microcentrifuge tube.
      2. Add 50 µL of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Then centrifuge at 16,000 × g for 1 minute.
      3. Discard the Minicolumn and store the microcentrifuge tube holding the DNA at 4°C or –20°C.
    • Gibson Assembly ﹀

      The Gibson Assembly is based on the protocol provided by New England Biolabs.


      1. Thaw 10 µL of 2X Gibson Assembly Master Mix on ice.
      2. Add DNA vector and insert. The recommended ratio of mol vector to mol insert is 1:3. The volume of vector and insert together should not exceed 10 µL.
        Component Volume (µL)
        Gibson Assembly Master Mix 2X (NEB) 10
        Vector X
        Insert Y
        MilliQ 10 - X - Y
      3. Incubate the assembly reaction at 50°C for 1 hour.
    • Ligation ﹀
      1. Thaw the ligase buffer on ice.
      2. Add the following components together:
        Component Volume (µL)
        10X ligase buffer 2
        T4 DNA ligase 1
        DNA vector X (~100 ng)
        DNA fragment Y*
        MilliQ 17 - X - Y

        3. * The recommended vector to insert ratio is 1:3.

      3. Incubate at 4°C for at least 1 hour. Optimally, incubate overnight at 4°C.
    • MoClo ﹀

      Modular cloning based on the Golden Gate Cloning protocol.


      Pipetting scheme for MoClo level 0:

      Component Volume (µL)
      DNA insert (40pmol) 0.5
      Vector (40pmol) 0.5
      T4 DNA Ligase buffer (NEB) 1
      T4 DNA Ligase (NEB) 0.5
      BpiI 0.5
      Nuclease Free Water 7
      Total 10

      Pipetting scheme for MoClo level 1 and 2:

      Component Volume (µL)
      Each DNA insert (40pmol) 0.5
      T4 DNA Ligase buffer (NEB) 1
      T4 DNA Ligase (NEB) 0.5
      Level 1: BsaI V2 (NEB)
      Level 2:BpiI       		0.5
      Nuclease Free Water Up to 10
      Total 10

      Place the PCR tube in a thermocycler with the following protocol for all levels:

      Step Temperature (°C) Time
      1 37 2min
      2 16 5 min
      Repeat steps 1 and 2 50x
      3 50 10min
      4 80 10min

    • Nanodrop DNA quantification ﹀
      1. Use a nanodrop spectrometer to quantify the DNA amount and purity in a DNA purification or extraction sample.
      2. Adjust the settings to measure dsDNA at 260nm and impurities at 230 nm and 280 nm.

      Note: DNA should have a 260/280 between 1.8-2.0. The sample is contaminated if the value is below 1.8.

    • PCR ﹀
      • Colony PCR (GoTaq) ﹀
        1. Add the following components for a colony PCR in a PCR tube:

          2. Component Volume (µL) Final concentration
          Gotaq 5X buffer 10 1x
          10 mM dNTP’s 1 200µM
          Forward Primer 1 200nM
          Reverse Primer 1 200nM
          Colony*
          GoTaq Polymerase (5U/µL) 0.2 20U/mL
          Nuclease Free Water 36.8
          Total 50

          *Pick a colony under aseptic conditions and resuspend it in milliQ water.


        2. Place the PCR tube in a thermocycler with the following protocol:
          3. Step Temperature (°C) Time
          Initial Denaturation 98 150sec
          Denaturation 94 60sec
          Annealing Tannealing* 60sec
          Extension 72 1min/kb DNA
          Repeat 29x the steps denaturation to extension
          Final Extension 72 5 min
          Hold 4 ∞

          * Tannealing: The annealing temperature is dependent on the melting temperature (Tm) of the primers. Tannealing=Tm - 5°C.

        3. Check the PCR product on an agarose gel (see DNA electrophoresis protocol)
      • Phusion PCR ﹀
        1. Add the following components for a Phusion PCR in a PCR tube:

          2. Component Volume (µL) Final concentration
          5X Phusion HF Buffer 10 1x
          10 mM dNTP’s 1 200µM
          Forward Primer 1 200nM
          Reverse Primer 1 200nM
          Template DNA (10ng) X
          Phusion Polymerase 0.5 20U/mL
          Nuclease Free Water 36.5 - X
          Total 50

        2. Place the PCR tube in a thermocycler with the following protocol:
          3. Step Temperature (°C) Time
          Initial Denaturation 98 30sec
          Denaturation 98 10sec
          Annealing Tannealing* 15sec
          Extension 72 30sec/kb DNA
          Repeat 29x the steps denaturation to extension
          Final Extension 72 5 min
          Hold 20 ∞

          * Tannealing: The annealing temperature is dependent on the melting temperature (Tm) of the primers. Tannealing=Tm - 5°C.

        3. Check the PCR product on an agarose gel (see DNA electrophoresis protocol)
      • KOD Extreme PCR ﹀
        1. Add the following components for a Kod Extreme PCR in a PCR tube:

          2. Component Volume (µL) Final concentration
          2x Kod Xtreme Buffer 2 1x
          10 mM dNTP’s 2 400µM
          Forward Primer 1.5 300nM
          Reverse Primer 1.5 300nM
          Template DNA (100ng) X
          Kod Polymerase 1
          Nuclease Free Water 36.5 - X
          Total 50

        2. Place the PCR tube in a thermocycler with the following protocol:
          3. Step Temperature (°C) Time
          Initial Denaturation 94 2min
          Denaturation 98 10sec
          Annealing 68 30sec
          Extension 68 4min
          Repeat 29x the steps denaturation to extension
          Final Extension 68 7min
          Hold 12 ∞
        3. Check the PCR product on an agarose gel (see DNA electrophoresis protocol)
    • PCR Clean-Up ﹀

      Protocol for PCR Clean-Up using Promega Wizard SV PCR Clean-Up System:



      Processing PCR Amplifications:

      1. Add an equal volume of Membrane Binding Solution to the PCR amplification.

      Binding of DNA

      1. Insert SV Minicolumn into Collection Tube.
      2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
      3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.

      Washing

      1. Add 700µl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
      2. Repeat Step 4 with 500µl Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.
      3. Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.

      Elution

      1. Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube.
      2. Add 50µl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute.
      3. Discard Minicolumn and store DNA at 4°C or –20°C.
    • Plasmid Isolation ﹀

      Protocol for plasmid isolation using Promega PureYield MiniPrep Kit:

      Prepare lysate:

      1. Pipette 1.5ml of the bacterial culture in an eppendorf tube and centrifuge culture for 30 seconds at maximum speed in a microcentrifuge.
      2. Discard the supernatant.
      3. Repeat Steps 1 and 2 until the entire inoculum volume has been spun down.
      4. Add 600µl of MilliQ water to the cell pellet, and resuspend completely.
      5. Add 100µl of Cell Lysis Buffer, and mix by inverting the tube 6 times. The solution should change from opaque to clear blue, indicating complete lysis.
        6. Note: if you have more sample volume, split it. This will help complete this step quickly.
        Note: Proceed to Step 6 within 2 minutes. Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, process samples in groups of ten or less. Continue with the next set of ten samples after the first set has been neutralized and mixed thoroughly.
      6. Add 350µl of cold (4–8°C) Neutralization Solution, and mix thoroughly by inverting the tube. The sample will turn yellow when neutralization is complete, and a yellow precipitate will form. Invert the sample an additional 3 times to ensure complete neutralization.
      7. Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
      8. Transfer the supernatant (~900µl) to a PureYield™ Minicolumn. Do not disturb the cell debris pellet. For maximum yield, transfer the supernatant with a pipette.
      9. Place the minicolumn into a PureYield™ Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds or 30 seconds.
      10. Discard the flow through, and place the minicolumn into the same PureYield™ Collection Tube.

      Wash:

      1. Add 200µl of Endotoxin Removal Wash to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 seconds. It is not necessary to empty the PureYield™ Collection Tube.
      2. Add 400µl of Column Wash Solution to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds.

      Elute:

      1. Transfer the minicolumn to a clean 1.5ml microcentrifuge tube, then add 30µl of nuclease free water at neutral pH directly to the minicolumn matrix. Let stand for 1 minute at room temperature.
        2. Notes: 1. Elution buffer can also be used, but it can interfere with future cloning steps. 2. For large plasmids (>10kb), warm the Elution Buffer to 50ºC prior to elution, and increase elution volume to 50µl. Also incubate the column at room temperature (22–25°C) for 5–10 minutes before proceeding to Step 14.
      2. Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute the plasmid DNA. Cap the microcentrifuge tube, and store eluted plasmid DNA at –20°C.
      3. Follow the nanodrop quantification protocol to determine the purity and yield of your plasmid isolation.
    • Restriction Digestion ﹀
      1. Choose the desired enzyme and its required buffer. The CutSmart buffer 10X is used for most NEB restriction enzymes.
      2. Add the following components together:
        Component Volume (µL)
        10X CutSmart buffer (NEB) 2
        Fragment (~1-2μg) X
        Restriction Enzyme 1/enzyme
        MilliQ 18 - X - added reaction enzyme
      3. Incubate for 4 hours at 37 °C.
      4. Inactivate the restriction enzyme by heating to 65 °C for 10 minutes.
  • Strain Specific ﹀
    • Bacillus Subtillis ﹀
      • Electrocompetent Cells Preparation ﹀
        1. Dilute an overnight culture of Bacillus subtilis 16-fold in growth medium and grow at 37 °C to an O.D.600 of 0.85-0.95.
        2. Cool the cells on ice-water for 10 min. and harvest by centrifugation at 4°C and 5000 x g for 5 min.
        3. Wash cells four times in ice-cold electroporation medium.
        4. Suspend the cells in 1/40 of the culture volume of the electroporation solution with a cell concentration of 1-1.3 x 1010 cfu/mL.
        5. The competent cells can be stored at –80 °C until use with some decrease in transformation efficiency.
      • Transformation using electroporation ﹀
        1. Add 1 µl (50 ng/µl) plasmid DNA to 60 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette. Incubate for 1-1.5 min.
        2. Wipe moisture from the cuvette and insert the cuvette into the device.
        3. Electroporation:
          4. mode Prokaryotes "O"
          Voltage (V) 2,100 V
          Time constant (τ) 5 ms
          No. of pulses (n) 1
        4. Immediately add 1 ml outgrowth medium and incubate for 3 h at 37 °C.
        5. Plate onto selective LB agar plates and incubate overnight at 37 °C.
    • Escherichia coli ﹀
      • Chemically Competent Cells Preparation ﹀

        The protocol for chemical competent cells has a duration of three days, starting from the preparation of the reagents. Throughout the protocol, it is mandatory to work under aseptic conditions to prevent contamination risks as much as possible.

          Day 1 (preparation):
        1. Prepare the necessary reagents and sterilize the reagents and centrifuge bottles.
          • 1 L Luria Broth (LB) medium
          • 10 mL Luria Broth (LB) medium
          • 400 mL 100mM CaCl2
          • 20 mL 100 mM CaCl2+ 15% glycerol
        2. Store the CaCl2 and the CaCl2 + 15% glycerol stock solution at 4°C for at least 24 h.
        3. Take the strain of interest from the -80°C and store it on ice.
        4. Streak out the desired E. coli strain on LB plate with antibiotic selection marker and incubate at 37°C overnight.

          5. Day 2 (preparation):
        5. Inoculate one single colony from the overnight grown plate in 10 mL LB medium with antibiotic selection marker overnight at 37°C while shaking at 200 rpm.

          6. Day 3:
        6. Inoculate 10 mL from the overnight grown liquid cell culture in 1 L of LB medium with antibiotic selection marker and grow the inoculated medium at 37°C while shaking at 200 rpm until the OD600 is between 0.4 and 0.6.The OD is measured the first time after 1.5 hours after inoculation. (Important: the OD should not rise above 0.6 in order to obtain good competent cells)

          7. Important notes:
          • From this point be very careful with your cells. The cells are very fragile.
          • Everything that touches the cells should not be warmer than 4°C.
          • Keep the cells always on ice. Thus also resuspension steps needs to be performed on ice.
          • Work clean and fast, the cells heat up very quickly which influence the quality of the competent cells a lot.
        7. Split the 1 L LB medium with cells into the centrifuge cylinders by weighing them in order to have equal weight distribution during centrifuging. If necessary prepare a counterweight for in the centrifuge.
        8. Harvest the cells by centrifugation for 5 minutes at 4000 rpm at 4°C and resuspent all of the pellets carefully in 200 mL ice cold CaCl2. Use an electronic pipet to resuspend the pellet.
        9. Incubate the resuspended cells for 20 minutes on ice.
        10. Harvest the resuspended cells by centrifugation for 5 minutes at 3000 rpm at 4°C, and resuspent the new pellet gently in 100 mL ice cold CaCl2. Use an electronic pipet to resuspend the pellet.
        11. Incubate the resuspended cells for 1 hour on ice. In the meantime mark approximately 75 1.5 mL Eppendorf tubes and store them on ice.
        12. Harvest the resuspended cells by centrifugation for 5 minutes at 3000 rpm at 4°C, and resuspent the new pellet gently in 10 mL ice cold CaCl2 + 15% glycerol. Use an electronic pipet to resuspend the pellet.
        13. Aliquot 50 μL into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. (Since the cells are very fragile, cut the pipettips in order to reduce the stress on the cells.)
        14. Store frozen cells in the -80°C freezer.
        15. Immediately perform a heat shock transformation to determine the transformation efficiency.
      • Glycerol Stock ﹀
        1. Incubate ~50 µl cells in 5 ml LB and selection marker overnight.
        2. Pipette 1.5 ml in an eppendorf tube and spin down the bacterial culture.
        3. Discard the supernatant.
        4. Repeat step 2 and 3
        5. Resuspend the pellet in 0.9 ml liquid bacterial culture.
        6. Add the liquid bacterial culture from eppendorf tube to cryotube.
        7. Add 0.9 ml 50% glycerol to the cryotube and mix well.
        8. Store at -80°C.
      • LB Agar preparation ﹀
        1. Dissolve 14.24g LB agar in 400 mL of milli-Q.
        2. Autoclave the LB agar solution.
      • Liquid Broth (LB) Medium Preparation ﹀
        1. Dissolve 20 g/L Luria Broth (LB) powder in water as described in the instructions provided by the manufacturer.
        2. Autoclave the medium at 121°C to heat sterilize.
        3. Store the sterilised LB medium at room temperature.
      • Heat Shock Transformation ﹀
        1. Thaw 50 µL of chemical competent cells on ice for 10 minutes.
        2. Add 0.1- 10 ng plasmid DNA or 20 - 50 ng ligate to the competent cells. incubate on ice for 30 minutes.
        3. Heat shock 45 seconds at 42°C.
        4. Incubate 2 minutes on ice.
        5. Add 1 mL LB medium, mix gently.
        6. Incubate 1 hour at 37°C.
        7. Prepare agar plate with the right corresponding antibiotic marker. 25 mL agar with 200 µL of antibiotic stock solution.
        8. Plate 100 µL of cell suspension on the agar plate and grow it overnight at 37°C.
    • Pseudomonas Putida ﹀
      • Electrocompetent Cells Preparation ﹀
        1. Inoculate 50 ml standard-1 medium with 7 ml of a fresh overnight culture of Pseudomonas putida. Grow cells at 30 °C to a density of O.D. of 0.8.
        2. Harvest by centrifugation.
        3. Wash twice with 50 ml ice-cold glycerol, centrifuge.
        4. Resuspend cells in 0.8 ml ice-cold glycerol, keep on ice.
      • Transformation using electroporation ﹀
        1. Add 4 µl plasmid DNA (1 µg) to 40 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette.
        2. Wipe moisture from the cuvette and insert the cuvette into the device.
        3. Electroporation:
          4. mode Prokaryotes "O"
          Voltage (V) 2,400 V
          Time constant (τ) 5 ms
        4. Immediately add 1 ml standard 1-medium. Incubate 2 hours at 30 °C.
        5. Plate cells on selective plates.
    • Vibrio Natriegens ﹀
      • Electrocompetent Cells Preparation ﹀
        1. Grow a 10mL culture of V.natriegens cells in BHIv2 overnight.
        2. Inoculate 100mL BHIv2 with 1mL of overnight culture.
        3. Place in orbital shaker at 37C at 200 rpm until OD600=0.5.
        4. Separate culture into two 50mL falcon tubes and place on ice for 15 min.
        5. Pellet cells at 6500 rpm for 20 min at 4C.
        6. Resuspend cells in 5 mL(each tube, 10 mL total) of electroporation buffer (680 mM sucrose, 7 mM K2HPO4, pH 7).
        7. Fill tube to ~35mL of electroporation buffer and invert gently to mix.
        8. Centrifuge cells at 6500 rpm for 15 min at 4C.
        9. Decant supernatant with a pipette.
        10. Wash the cells with electroporation buffer as described above a total of three times.
        11. Wash the cells with electroporation buffer as described above a total of three times.
        12. After the final wash, resuspend cells in electroporation buffer as to have OD600=16.
        13. Aliquot cells at 100 uL each tube and store in -80°C
      • Transformation using electroporation ﹀

          Transformation in Vibrio natriegens SGI-DNA Vmax manual

        1. For each transformation reaction,
          • Thaw one vial of Electrocompetent cells on ice for 5 minutes.
          • Warm one vial of BHIv2 (BHI with v2 salt: 204 mM NaCl, 4.2 mM KCl, and 23.14 mM MgCl2) to room temperature (20°C to 24°C).
          • Pre-chill a 0.1-cm electroporation cuvette on ice.
        2. As soon as the electrocompetent cells are thawed, add 10 ng – 100 ng of plasmid DNA (in TE buffer or water only) to the competent cells on ice. Briefly mix by flicking tubes. Note: Add no more than 2 μL of plasmid DNA to the competent cells. Concentrate the plasmid DNA if necessary.
        3. Incubate the DNA and cell mixture on ice for 3–5 minutes.
        4. During incubation, set the following electroporation parameters: 900 V, 25 uF, 200 Ohms, 0.1-cm cuvette. If further optimization is desired, voltage changes in the range of +/- 200V in 50 V increments can be used. Note: These are the optimized electroporation settings for the Gene Pulser® XcellTM Microbial System using a cuvette with a 0.1-cm gap. If other instruments and/or cuvettes are used, you may need to optimize the conditions for best results.
        5. Transfer cells to a chilled cuvette using a pipette. Verify that the cells completely occupy the bottom of the cuvette and that no air remains between walls of the cuvette and the cells.
        6. Transfer the cuvette to the shock pod and perform electroporation.
          • Note: Time constants typically range from 3.3–3.7 ms.
        7. Add 500 uL of BHIv2 recovery media to the cuvette immediately after pulse.
        8. Transfor the BHIv2 media and cell mixture to an Eppendorf tube and place in an orbital shaker (250 rpm) at 30°C for 2 hours at to allow recovery of cells and expression of antibiotic resistance marker.
          Note: Recovery can be performed at 37°C unless kanamycin is used for selection.
        9. During cell recovery, pre-warm LB-Miller agar plates prepared with antibiotics at the appropriate incubation temperature.

        Plating notes
        • We recommend incubating plates at 25°C to 30°C overnight.
        • For plates containing kanamycin, plates must be incubated at 25°C to 30°C.
        • Colonies typically appear after 12–16 hours at 25°C to 30°C and after ~8 hours at 37°C.
        • Store agar plates with established VmaxTM Express at room temperature for up to 72 hours. Due to contact inhibition single colonies will remain well-isolated.
        • Do not store VmaxTM agar plates at 2–8°C as this can adversely impact propagation and growth.
        • For long-term storage, we recommend preparing glycerol stocks with 25% glycerol and storing at −80°C.

        Antibiotic Solid media Liquid culture
        Ampicillin 2-50 µg/mL 2-25 µg/mL
        Kanamycin 100 µg/mL 200 µg/mL
        Tetracycline 2.5 µg/mL
        Chloramphenicol 5–12.5 μg/mL 12.5–25 μg/mL