Polymerase Chain Reaction (PCR)

Owned by Lan Nguyen
Jun 21, 2021
  1. Wear gloves, and change them often!
  2. Sign up for the thermal cycler.
  3. Put pipet tips, PCR water, and PCR tubes in UV crosslinker for 5 minutes.
  4. Get a bucket of ice.
  5. Wipe down bench and pipets with ethanol. This is helping to ensure a clean environment for PCR, so no stray organisms will get into your reactions.

  6. Calculate the volumes needed for a 50 μL reaction. Then, calculate the volumes required for the number of reactions you are running plus a little extra.  I usually prepare an extra third of a reaction.  For the kit (PCR Master; Roche) and primers we are using today, the following volumes are appropriate:

    ComponentVolume (µL) - 1 sampleVolume (µL) - 7.3 samples

    PCR Master Mix

    25

    182.5

    8f (forward primer)

    0.5

    3.7

    926r (reverse primer)

    1

    7.3

    PCR water

    22.5

    164.3

    DNA sample/pure culture

    1


    Total Volume50357.8

    **Other kits give you more flexibility with concentrations, and you can make up the Master Mix as you choose. This kit offers simplicity since the Master Mix is pre-mixed.

  7. Take the Master Mix, primers, and DNA sample from the freezer.  Keep them on ice until you are ready to use them.  When you are ready to dispense, warm each reagent in your hands.

  8. Add the Master Mix, primers, and PCR water to a single PCR tube.  Aspirate to mix.  Dispense 49 μL to the PCR tubes.  Keep these on ice.

  9. Always run positive and negative controls with your PCR reactions.

    1. Positive control - DNA that you know will amplify.  I often use a pure bacterial culture.  To the PCR tube, add 1 μL of a liquid culture or a small scraping from a plated culture.
    2. Negative control - No DNA template. To the PCR tube, add 1 μL of PCR water.
  10. Add your sample DNA to the remaining tubes. Add 1 μL of DNA to the PCR tube.
  11. Now you should have 50 μL in each PCR tube. Aspirate to mix them.  Centrifuge briefly (few seconds) to get all the liquid to the bottom of the tube.
  12. Turn the thermal cycler on. It will go through a self-test.
  13. Put the tubes into the thermal cycler. To have even heating in the cycler, I like to spread the tubes out instead of clumping them together.
  14. Close the lid on the cycler and select the desired program. We will be using the Liu protocol in the Mary Jo folder.  This protocol takes about 3.5 hours to complete.
  15. While the reactions are cycling, you can prepare a gel to look at the products. Prepare a 1% agarose gel in 1x TAE (i.e. 1 g agarose in 100 mL of TAE).  Melt in the microwave.  Let it cool for 5 minutes, and then pour it into a gel caster with a comb.  It will take about 15-20 minutes to solidify.
  16. When you are ready to run the gel, make sure that the gel is covered in the electrophoretic chamber with 1x TAE.
  17. Tape down a strip of parafilm. Pipet one 2-μL volume of 6x loading buffer for each sample to be run.  To each of these, add 10 μL of PCR product.  Aspirate to mix.
  18. In the first lane, add 1 kb ladder. (The 1 kb ladder already has loading buffer added to it.)  In the next lanes, add the controls and samples.
  19. Run the gel at 70 mA (constant mA). Check its progress after 45 minutes-1 hour.
  20. Stain with ethidium bromide (EtBr) for about 20 minutes. Always wear gloves when dealing with EtBr since it is a mutagen.  I like to wear two sets of gloves.  Dispose of your gloves and gels with EtBr in the designated receptacle.  DO NOT TOUCH COMMON AREAS WITH EtBr-CONTAMINATED GLOVES.  This means that you should take off these gloves when you want to use a pipet, open a drawer, open the waste receptacle, etc.
  21. Look at the gel under UV light. You should see a band in the positive control and sample lanes and nothing in the negative control lane.
  22. Store the PCR product at -20o


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