Amplifying a 10kb fragment from a 13kb plasmid using Q5 polymerase?
Has anyone successfully amplified a 10kb fragment from a plasmid (mine is 13kb) using the Q5 High-Fidelity polymerase?
I tried using an extension time of 5 minutes at 72C. My gel shows that my plasmid is intact and runs at >10kb, but the PCR has only a faint smear from ~3kb to ~500bp. My primers bind specifically after checking on SnapGene, and another PCR I ran in parallel for a 1kb fragment worked, so the reagents are okay. Has anyone had any luck doing this, and what conditions worked?
Thanks!
Posted Jul 10, 2026, 6:03 PM UTC
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