Field of Science

DAMN! Complete PCR failure!

Yesterday I ran a PCR amplification using DNAs from single colonies of 7 different A. pleuropneumonia isolates, and got absolutely no DNA fragments from any of them.

This amplification worked fine last time.  Can I figure out what went wrong?

  • I checked the run record of the PCR machine - it looks fine.
  • I checked the freezer box with the tubes of dNTP stock, 5X buffer, and Q5 polymerase, to be sure I hadn't picked up a wrong tube.
  • I checked my notes, to be sure I hadn't left out any component of the reaction mix.  I'd checked off each reagent as I added it, and the final volume was as expected.
  • I checked the 'F' and 'R' primer tubes (in another freezer box) to make sure I'd used the correct ones.  I'd made up more of the 10 mM dilution stock, so I also checked that I'd used the right tubes of the more-concentrated 100 mM stock to do this.  I even checked the remaining volumes in the two primer tubes - if I'd added one primer twice and not the other these volumes should differ by about 17 µl, but they're within a few µl.
  • I prepped the colony DNAs slightly differently.  Last time (prep 1) I put a whole colony into 100 µl of medium, then diluted 5 µl of that into 45 µl water and heated to 98 °C for 10 min to lyse the cells and free their DNA.  This time (prep 2) I put part of a colony into 100 µl water, heated that, and then pelleted out any cell debris.  Both times 1 used 1 µl of the heated sample.
What could I try now?
  • Use leftover Prep 1 colony DNA as template
  • Vortex the Prep 2 colony DNA tubes
  • Use as template purified DNA from lab stocks
  • Use a different pair of primers (the Spec-cassette ones worked well last time)
  • Repeat with the same reagents and template I used this time
  • Make fresh colony DNA preps
  • Make proper DNA stocks to use as templates
  • Prep 2 14-1 colony DNA, Spec primers
  • Vortexed Prep 2 14-1 colony DNA, F & R primers
  • Prep 1 14-1 colony DNA, F & R primers
  • Prep 1 14-1 colony DNA, Spec primers
  • 1/100 dilution of lab-stock DNA, F & R primers


    1. Just do it again and accept something went wrong?

    2. Prepare new water / buffer for the dilutions and other steps. "Bad water" is pretty vague, but I've had good luck in the past improving PCR success by tossing all of the autoclaved water I was using and getting new water.


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