Colony-based PCR

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The method below is based on “Effect of Diet on Bean Beetle Microbial Communities” by Cole et al. to be published in Tested Studies for Laboratory Teaching, Proceedings of the Association for Biology Laboratory Education, Volume 39. Vinny Ciavetta at Emory University helped to optimize the protocol.

The order to follow is:

  1. Assemble the PCR master mix on ice. (You might prepare master mix for students.)
  2. Aliquot 15 µL of the PCR master mix to 6 tubes of a labeled PCR strip (leave the other 2 tubes empty).
  3. Pick a bacterial colony.
  4. Mix the bacterial colony in water (henceforth, colony suspension).
  5. Add 10 µL of the colony suspension, positive control DNA, or water to the correct PCR tube that contains the PCR master mix.

Specific instructions for each step:

  1. Assemble the PCR master mix on ice.
    1. Be sure all reagents thaw completely before starting. Keep reagents on ice once thawed.  Vortex and then spin each reagent briefly (for small tube spin inside of an empty 1.5mL tube).  Place each reagent back on ice.
    2. In a 1.5 mL microcentrifuge tube, add reagents in the order listed in the table below. Check-off as you go to be sure all reagents are added.
    3. After all reagents are added, cap the tube, vortex briefly to mix, spin briefly to force the liquid to the bottom of the tube, and return the tube to ice.

    2.  

    Reagent 1X 7X Check
    dH2O 5.75 40.25
    10X PCR Buffer 2.5 17.5
    50X dNTPs 0.5 3.5
    10X Forward Primer (27F) 2.5 17.5
    10X Reverse Primer (1429R) 2.5 17.5
    Taq polymerase 1.25 8.75
    Colony suspension 10.0 Add separately to each PCR tube
    total 25 105 N/A

    2uM forward primer – 27F (5′ – AGA GTT TGA TCC TGG CTC AG)
    2uM reverse primer – 1492R (5′ – GGT TAC CTT GTT ACG ACT T)

     

  2. Aliquot 15 µL of the PCR master mix to 6 tubes of a labeled PCR strip, on ice.
    1. Label the PCR strips. When you get the DNA sequencing results, you need to have all the information about the colony you sequenced. In your notebook, write down the order of reactions on your PCR strip including – plate type and colony morphology. Also note the location of your positive and negative controls.
    2. Add 15 µL of the PCR master mix to the bottom of each tube.
    3. Cap the tubes and keep them on ice.
  3. Pick a bacterial colony.
    1. Get 4 sterile 1.5 mL microcentrifuge tubes and label each.
    2. Add 100 µL of sterile dH2O to each tube.
    3. Set a P20 pipetteman to 10 µL.
    4. Attach a sterile yellow tip to the end of the P20 pipetteman.
    5. Gently touch the pipette tip to a well-separated colony from one of the bacterial growth plates. (Bacteria will stick to the pipette tip.) Do not scoop a glob of bacteria.
  4. Mix the bacterial colony in water (henceforth, colony suspension).
    1. Submerge the pipette tip in the appropriate tube of 100µL water and pipette up and down vigorously for about 5 seconds to make the colony suspension.
  5. Add 10 µL of the colony suspension to a tube that contains the PCR master mix.
    1. Using the same pipette tip that was used for picking and mixing, transfer 10 µL of the colony suspension to the master mix in the appropriate PCR tube.
    2. Pipette up and down 3 times to be sure the colony suspension and PCR master mix are well-mixed.
    3. Keep the PCR strip on ice.
    4. Go back to step 3d and repeat until one colony from each plate has been picked, suspended, and the suspension added to the correct PCR tube.
    5. For the positive control, add 10 µL of the positive control DNA to the PCR tube labeled for positive control. For the negative control, add 10 µL of sterile water to the PCR tube labeled for negative control.
    6. Cap the PCR strip so all tubes are sealed. Keep your assembled reactions on ice.
    7. Tell the laboratory instructor you are finished assembling your PCR reactions.

    6.  

    PCR Program

    95ºC, 10 min (purpose is to help disrupt bacterial cell walls/membranes to release dna)

    Then 36 cycles of:

    95ºC, 30 sec

    55ºC, 30 sec

    72ºC, 1.5 min

    Lastly,

    72ºC, 4 min,

    4 ºC, hold

    Visualizing and sequencing of DNA

    1. Pour an agarose gel
      1. Assemble your gel box and comb – make sure gaskets are in place
      2. Bring your agarose to the instructor to have 7.5uL Sybr Green added
      3. Swirl to mix well
      4. Pour in your gel box
      5. Pop any bubbles
    2. Prepare your samples
      1. First mix 2uL PCR reaction with 198uL water (save this for later sequencing)
      2. Mix 7uL 5X DNA loading dye to remaining PCR reaction
    3. Load gel
      1. Load 7uL premixed DNA ladder into the leftmost lane
      2. Load 20µL of your prepared samples into lanes skipping one lane between each sample
    4. Run your gel at ~135v until dye front is more than halfway through the gel
    5. Visualize your PCR reactions using the UV box
    6. Successful PCR reactions should load 10µL of your reserved for sequencing sample into your assigned well in the 96-well plate
    7. Add sequencing primers and send the plate out for sequencing. We use Eurofins for sequencing (https://www.eurofinsgenomics.com/en/home.aspx).