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