Where does my primer anneal?

[virpericulosus]

This seems like a dumb question, but I haven't been able to figure it out.

I sequenced hemoglobin genes using primers I found online/literature. I found the mutation I was looking for, but I've tried locating the primer annealing site on NCBI and I can't figure out where it would bind.

The gene is HBA2. The primers I used for exon 3 (where the mutation is) were:

F: gaggatcacgcgggttg
R: caggaaacagctatgacc (M13 reverse primer)

Any routes besides NCBI people can suggest? I have the sequencing file that shows the sequencing starting... but I don't know if that really shows where the primer annealed.

[dpryan]

The forward primer maps to the second intron of both hba1 and hba2, which seems reasonable. You're not going to get good genomic DNA amplification with an M13 reverse primer. Are you actually sequencing genomic DNA or did you or someone else perform some subcloning?

[virpericulosus]

We first used specific pcr primers to hba1 and hba2 and then sequenced using 3 pairs of primers.