Team:UrbanTundra Edmonton/Protocals


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Preparing Lysogeny Broth

  • Spray working surface with 10% bleach.
  • Place the plastic tray inside the scale and tare.
  • Weigh the appropriate mass of culture medium.
  • Using a graduated cylinder, measure and transfer the required volume of Milli-Q water to the medium bottle.
  • Deposit the media inside the bottle and shake vigorously to dissolve.
  • Autoclave the contents of the medium bottle.
  • Allow media to cool and store at room temperature.

    • Preparing Agar Plates

    1. Spray working surface with 10% bleach.
    2. Place the plastic tray inside the scale and tare.
    3. Weigh the appropriate mass of culture medium.
    4. Using a graduated cylinder, measure and transfer the required volume of Milli-Q water to the medium bottle.
    5. Deposit the media inside the bottle and shake vigorously to dissolve.
    6. Weigh the appropriate amount of agar, usually 15g/L.
    7. Deposit the agar into the bottle.
    8. Autoclave the contents of the medium bottle.
    9. Spray the bag of Petri dishes with 10% bleach and transfer the bag to the sterile work station.
    10. Remove the plates from the bag, placing them in stacks with their lids facing upwards.
    11. Transfer the recently autoclaved media bottle to the sterile work station.
    12. Add the required volume of antibiotic in proportion to the volume of media
      1. For kanamycin, 1 μL of 1000x kanamycin stock solution is added for every 1 mL of LB.
      2. For chloramphenicol, 100 μL of 1000x chloramphenicol stock solution is pipetted for every 100 mL of LB.
    13. Take a plate from the stack, remove the lid and place its top facing upwards. Then, place the plate with its bottom supported by the lid.
    14. Open the lid of the medium bottle and pour the LB agar medium onto plate until roughly ¾ of the bottom of the plate is covered.
    15. Swirl smoothly each plate to distribute the media over the entire plate.
    16. Once plate has been covered with media, set the plate off to side and repeat the procedure with a new plate until all plates contain media. It is important to work as efficiently as possible to pour the LB agar media into the petri dishes before the media solidifies in the bottle.
    17. Allow time for the plates to solidify.
    18. Once the plates are dry, place the lids back on and stack the plates with the lids facing upwards.
    19. Place the plates in the original bag with the agar side upwards.
    20. Tightly secure the opening of the bag with tape, and label the bag with the date, the type of media and the antibiotic added.
    21. Store the plates at room temperature.

    Autoclave

    1. Seal the lid of the media bottle with autoclave tape.
    2. Place in the autoclave, following the instructions/standards of your lab and autoclave.
    3. Check if the diagonal lines on the autoclave tape turned black before using.

    Inoculation of Growing Cultures in Liquid Media

    1. Spray working surface with 10% bleach.
    2. Add the required volume of antibiotic in proportion to the volume of Lysogeny Broth (LB).
      1. For kanamycin, 1 μL of 1000x kanamycin stock solution is added for every 1 mL of LB.
      2. For chloramphenicol, 100 μL of 1000x chloramphenicol stock solution is pipetted for every 100 mL of LB.
    3. Pipette 5 mL of antibiotic-treated LB into every culture tube or flask.
    4. Open the petri dish and using the needle end of the inoculation loop, scrape the desired colony from the plate.
    5. Close the petri dish and vigorously mix the colony into the appropriate tube or flask. Dispose of the loop into the biohazard waste bag.
    6. Place the tubes or flasks at an angle in the shaker and incubate at 37°C overnight.

    Plating E. coli Cultures on Agar Plates

    1. Spray working surface with 10% bleach.
    2. Pipette 200 μL of culture to the appropriate antibiotic-treated Lysogeny Broth (LB) agar plate.
    3. Use a bacterial cell spreader to gently spread the culture on the plate. Be careful not to press onto the plate.
    4. Place the plate under the fume hood with the lid off until it is dry.
    5. Incubate the plates with the lid side down at 37°C overnight.

    Cloning to Expression Vector (CPB-38-441)

    1. Pipette 1 μL of CPB-38-441, 4 μL of Milli-Q water, and 5 μL of DNA insert to the microcentrifuge tube. Mix the contents gently.
    2. Add 1.1 μL of 10x NEB cutsmart buffer and 0.5 μL of BsaI-HF. Mix gently, and pulse down in a centrifuge if necessary.
    3. Incubate at 37°C for approximately 1 hour.
    4. BsaI-HF is heat killed in a thermocycler set at 65°C for 20 minutes.
    5. Pulse down the tube in a centrifuge.
    6. Pipette 8 μL of Milli-Q water, 2 μL of T4 DNA ligase buffer and 0.5 μL of DNA ligase to the tube. Mix gently and pulse down in a centrifuge.

    Digestion (BioBrick)

    1. Add 14 μL of Milli-Q water, 2 μL of 10x NEB cutsmart buffer, 2 μL of recombinant pSB1C3, 1 μL of EcoRI endonuclease and 1 μL of PstI endonuclease to a microcentrifuge tube.

    Chemical Transformation

    1. Begin heating the water bath to 42°C.
    2. Spray working surface with 10% bleach.
    3. Remove frozen chemically competent DH5α E. coli cells from -80°C freezer and thaw on ice.
    4. Pipette the appropriate volume of DNA to the tube of chemically competent cells. Stir the mixture gently with the pipette tip.
      1. If transforming cloned vector, add 1 μL of recombinant plasmid.
      2. If transforming intact plasmid, add 10 μL of the plasmid.
    5. Incubate tubes in an ice bucket for 30 minutes.
    6. Heat shock the tubes in a heating bath set at 42°C for 90 seconds.
    7. Transfer all the tubes in an ice bucket and let them sit for 5 minutes.
    8. Add 1 mL of Lysogeny Broth media to each tube.
    9. Incubate them for approximately 1 hour at 37°C.

    Miniprep

    1. Label six microcentrifuge tubes with the appropriate treatment group and sample number.
    2. Pipette 1 mL of overnight culture from the culture tubes into their corresponding microcentrifuge tubes and centrifuge at 13 000 rpm for 1 minute. Decant the supernatant. Repeat this step until  worth of culture has been centrifuged. If needed, pipette out the remaining liquid.
    3. Add 250 μL of Buffer P1 into each of the tubes and resuspend the pellet by vortexing.
    4. Add 250 μL of Buffer P2 into each of the tubes and invert the tubes 5-6 times.
    5. Add 350 μL of Buffer N3 into each of the tubes.
    6. Centrifuge all tubes at 13 000 rpm for 10 minutes.
    7. Label six QIAprep 2.0 spin column with the appropriate treatment group and sample number.
    8. Pipette a minimum of 800 μL of supernatant from each of the microcentrifuge tubes into their corresponding QIAprep 2.0 spin column.
    9. Centrifuge all the columns at 13 000 rpm for 1 minute.
    10. Decant flow through.
    11. Centrifuge all the columns at 13 000 rpm for 1 minute.
    12. Label six microcentrifuge tubes with the appropriate treatment group and sample number.
    13. Transfer columns to their corresponding microcentrifuge tubes.
    14. Add 100 μL of Buffer EB and let the solution sit for 1 minute.
    15. Centrifuge all the tubes at 13 000 rpm for 1 minute and discard the columns.

    Gel Electrophoresis

    1. Measure 30 mL of 1X TAE buffer using a graduated cylinder and deposit into a 250 mL Erlenmeyer flask.
    2. Weigh out 0.24g of agarose.
    3. Add to TAE buffer in Erlenmeyer flask. This will make a 0.8% gel.
    4. Microwave the flask to dissolve the agar and carefully swirl the flask to ensure that all of the agarose has dissolved.
    5. Pour the melted agarose into the gel casting tray and carefully insert the comb.
    6. Let the gel set for at least 20 minutes.
    7. Carefully remove the comb from the set gel.
    8. Place the casting tray into the gel box with the wells nearest to the black electrode (bottom of gel nearest to red electrode).
    9. Add 1X TAE buffer to the gel box to completely cover the gel. Pour enough buffer so that there is approximately 1 cm of buffer overtop of the gel.
    10. Prepare your samples for loading. Add 6X loading dye to each sample to a final concentration of 1X.
    11. Load 0.5-1 µl of ladder and 5-10µl of your sample into each well depending on the size of the well. Be careful not to stab the bottom of the well with the pipette tip.
    12. Close the lid for the gel box, matching the electrodes appropriately.
    13. Turn on the power supply and run at 80V for 40-90 minutes depending on the sample. Monitor the migration of the colored loading dye to ensure the samples do not run off the bottom.
    14. When the gel has finished running, turn off the power supply and remove the lid.
    15. Carefully remove the gel from the casting tray and place into the staining container of ethidium bromide.
    16. Let the gel stain in ethidium bromide for approximately 30 minutes.
    17. Transfer the gel to the platform in the gel doc. Close the cabinet door before turning on the UV light.
    18. Use the AlphaImager program to visualize the gel and capture an image.
    19. Turn off the UV light before opening the cabinet door.
    20. Dispose of the gel, buffer and any other ethidium bromide contaminated articles in the appropriate waste containers.

    Spectrophotometry (aka NanoDrop)

    1. Blank the NanoDrop twice with 2 μL of Buffer EB.
    2. Measure the 260/280 values and DNA concentration of 2 μL of each of the miniprep samples twice. Wipe the NanoDrop with Kimwipes after every measurement.

     

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