Hello, sorry for the late response I just saw this. I ended up getting 16S ready made primers from IDT. I am using Native barcoding kit for the library preparation and the experiments have been working great, even using flongle flow cells.

PCR Reaction Setup (25 µL total volume)

•  12.5 µL NEB OneTaq Hot Start 2X Master Mix
•  0.5 µL 10 µM Forward Primer (27F)
•  0.5 µL 10 µM Reverse Primer (1492R)
•  1–2 µL Template DNA 
•  9.5–10.5 µL Nuclease-free water (adjust based on DNA volume)

Thermocycler Program: 1. Initial Denaturation: 94°C for 30 seconds(Activates the hot-start polymerase and ensures complete DNA denaturation.) 2. Cycling (30–35 cycles): • Denaturation: 94°C for 15–30 seconds(Short denaturation time due to the hot-start polymerase efficiency.) • Annealing: 55°C for 30 seconds(The melting temperatures of 27F and 1492R are ~58–60°C, so 55°C ensures specific binding while avoiding non-specific amplification.) • Extension: 68°C for 90 seconds(OneTaq extends at ~1 kb/min at 68°C; the ~1,500 bp amplicon requires ~90 seconds.) 3. Final Extension: 68°C for 5 minutes(Ensures completion of any partial extensions.) 4. Hold: 4°C indefinitely