I am doing my undergrad thesis that requires me to do PCR-RFLP analysis on DNA that I extracted from some blood samples. Initially, I was using the Qiagen mastermix for the optimization of my entire process using just 6 samples out of 100 samples. I successfully completed my optimization step, and got my desired results for those 6 samples. Once I have the measurements of the entire process i.e the amount of mastermix, forward priner, reverse primer, DNA template, and nuclease-free water along with the optimum annealing temperature for my selected primer, I proceeded on to doing the exact same thing on the remaining 96 samples serially. But I started encountering an unexpected error. For some reason, I started getting a faint band in my negative control(which only has nuclease-free water and no DNA template). At first I thought any of the reagents were contaminated, so I conducted a series of PCR reactions where in one I would use a freshly new mastermix, another time a fresh made working solution of primers and also an intact nuclease free water. In every PCR, I am getting a band in my negative control. Today I also tried using a mastermix of an entirely different company i.e. Takara Bio, but got the same results. I am just frustrated at this point. Can anyone help me figure out what is happening? How can I troubleshoot this and complete my thesis!
Hello Mysha,
You can try dissembling and disinfecting the pipettes, if your using a hood for master mix preparation, you can also disinfect that with sodium hypochlorite, then ethanol then additional UV if the hood has the provisions for UV sterilization and hopefully use filtered tips during loading.
Hello Mysha
It's likely due to contamination. As Tevin mentioned, you should double-check to ensure your environment and equipment are sterile before conducting the PCR. Also, your nuclease-free water should be kept well before PCR as it can be contaminated as well
.
When you open your PCR tube after reaction, for instance when you need to load your PCR product on electrophoresis, it is believed, the PCR products can get into air aerosols and contaminate the next PCR reaction. Can you try to mix your PCR with new reagents, but also in a different laboratory, even a different building, and see if the incorrect product disappears ?
Otherwise, it is generally recommended to have different rooms for DNA isolation, mastermix mixing and loading the electrophoresis. Sometimes digestors are used in those rooms to suck the air away.
An expensive solution to prevent the *future* aerosol contamination is the use of dUTP instead of dTTP in the PCR mastermix. (Google the principle).
Hi!
This could be due to cross contamination.
Do through cleaning of your area and pipettes with either DNA Zap/ DNA away etc.
After that take wipe of each area and the pipette to establish the source of contamination.
Always use separate pipettes for making master mixes, addition of template and addition of positive control.