u/JaneDUT

Hi everyone,

I’m trying to linearize a 12.2 kb plasmid by PCR using NEB Q5 polymerase, but I’m getting no bands on the gel.

My PCR program is:

98°C 30 s
30 cycles of:
98°C 10 s
68°C 30 s
72°C 10 min 30 s
Final extension: 72°C 2 min
4°C hold

The annealing temperature was calculated using the NEB Tm calculator. The plasmid GC content is about 51%, so I don’t think GC enhancer is necessary.

I checked the reverse primer using IDT OligoAnalyzer. It predicts a hairpin with:

Hairpin Tm: 62.4°C
ΔG: −3.14 kcal/mol

But I think that's ok because ΔG is not so negative and a 68 annealing temp would disrupt the haripin structure.

I tried using 10 ng and 20 ng plasmid templates, but both failed. I haven’t gotten any visible PCR product.

I’m wondering what the most likely issue is. Could the reverse-primer hairpin still be a problem, or should I focus more on annealing temperature, template amount/quality, or long-PCR conditions?

Any suggestions for troubleshooting would be greatly appreciated. Thanks!