I am in a bit of a dilemma.
I am isolating soil genomic DNA and am using fungal specific primers to isolate certain families of fungi that I plan to clone and sequence. The soil genomic DNA mix that I extract is a mixture of all sorts of DNA and using fungal specific primers I hope to locate various fungi specific to those primers. In other words, there is not fixed product size that I am working with. The way these primers have been designed is through multiple sequence alignments of these fungi. These primers have been used before in other papers.
My reverse primer is (5' to 3') is say X and my forward primers for 4 classes of fungal specific primers are (5' to 3') A,B,C,D.
For each soil genomic DNA samples, I set-up 4 PCR reactions, A and X, B and X, C and X, D and X.
I had some very basic questions:
- When I BLAST my forward primer, I obtain results for various fungal species specific to those primers. They give me lengths and the starting point for that primer.
- When I BLAST the reverse primer, do I reverse complement it or reverse it or just keep it the way it is to obtain results ? This is a very basic question but I am confused !
After that, do I, for example, pick one of the BLAST matches common to BOTH the primers, search for my primer matches in THAT particular sequence and THEN estimate product size ? Even so, that's just one estimate. how do I get ballpark figures for all sorts of species for that particular family specific primer ?
Thanks a lot !