Hello,

If I understood correctly the small band is causing you problems. This happens in PCRs when the primers you use can hybridize elsewhere on the genome, leading to non-specific hybridizations.

If you need to recover just the band corresponding to your target gene, do a gel purification of your target band, then do sequencing to confirm that it is indeed your target sequence.

As the small band is weak (small pcr bands are usually stronger than longer products in the same reaction) and re amplification of the small band gives you some large band it looks like you have area of high GC or high AT in your sequence and some of your long pcr product is self annealing in a loop structure which is really the same thing and the same size as the longer product but runs quicker through the gel ( a lot like plasmid dna runs at different sizes). 3ng/ul seems anough to sequence so the sequence may be failing at the loop double starnded region.

If you run the pcr in presence of 5%dmso it should stop secondary structures from forming and give you a cleaner pcr and might indicate that secondary structures are causing this effect

If I understood correctly the small band is causing you problems. This happens in PCRs when the primers you use can hybridize elsewhere on the genome, leading to non-specific hybridizations. First check the Prime sequence with the genome of Enterococcus faecalis through the primer-BLAST. I think you used PCR reaction mixture with high concentration of MgCl2, and Gene specific Primer. May be you change the concentration of MgCl2, Primer and Anneling temperature increase 2 C.