I have a project asking to quantify a specific bacteria strain from a sample using qPCR. First I need to design one or more primers. Since I am new to primer design, I got several questions while designing the protocol.
- Should I use 16S hypervariable region as PCR template?
- Should I use Primer-BLAST as primer design tool? and should I used Genome database (reference assembly from selected organisms) as database for the design?
- If I got multiple hits, does it mean they are qualified as PCR primers?
- How many pairs of primer should I use to be sufficient to specifically target particular strain?
Can anyone has this kind of experience share some insight? Really appreciate!