Today we will give you a friendly advice.
Last month, Maithê and I (Paula) were making some reactions for real-time PCR. As usual, we planned the entire experiment with our advisor. We calculated all reagents that we would to use and we schematised the plate for the real-time PCR. Everything looked fine!
However, one of the necessary primers suddenly vanished when we were pipetting the plate!
This reagent was used just a few times, Maithê and I didn't know what was wrong. There should be plenty of primer left! We went to talk to our advisor and after some thought, the mystery was solved.
Solution: We followed the manufacturer's instructions!
Yes, that is right, the problem was follow the manufacturer's instructions.
Making a long story short, they recommend to use 50 uL final volume, but use 25 uL to calculate the number of reactions the kit can perform. Easy to compute, if you use the recommended volume, you will be able to do only half of the reactions they advertise.
At least, they recognised their mistake and are sending a new primer for replacement.
Lesson for the undergrad students: we can prevent these situations and alike by paying attention to the used reagents. We could (and should) have realised this primers was ending just by looking to the remaining volume. This would avoid bigger problems, such as the delay we are experiencing and waste of reagents.
The piece of advice? Do not trust manufacturer and pay close attention to the volume of all reagents you need.