Wondering if any of you have amplified whole mtDNA using long-range PCR? I'm having a small problem and would love to hear from someone who has done this before. So the organism I'm working with has a mtDNA genome of about 15 kbp. I designed primers which are close together (500 bp) with hopes they would do nearly the whole genome in one pcr (which has been done in my lab on other taxa using other primers). I wanted to be able to test the primers to make sure the priming sites were good before attempting the long range, so I also ordered the reverse complements of both so that I could amplify the 500 bp fragment between them, using standard pcr, as a test of their priming ability. So, the test of my primers worked wonderfully, so I'm sure the priming sites are good, but when I switch to long-range PCR I just get a smear. The DNA should be good, it was freshly extracted using a Qiagen kit and the samples were not stored (EtOH) long before extraction. So my question is, anybody have any protocol tweaking experience that worked? I'll add one thing, the smear doesn't have even a hint of a band and doesn't appear to be the kind that raising the annealing temperature would help. Thanks for any input you can provide.
P.S. Image is of 2 samples and a slightly overloaded hindi III ladder.