I'm performing site-directed mutagenesis using Agilent’s QuickChange kit. My goal is to introduce a single nucleotide substitution to change one amino acid. The primers were designed using Agilent’s online tool, and my plasmid is approximately 11 kb in size.

Here are the key details of my protocol:

• Strain: DH5α competent cells
• Antibiotic: Ampicillin
• DpnI digestion: 37 °C for 1 hour
• PCR elongation time: 6 minutes per cycle
• Plasmid size: ~11 kb

After transformation, I observed a few larger colonies (likely the main clones) surrounded by many satellite colonies. I picked several colonies—including some larger ones—for sequencing, but the results were messy and did not show the intended mutation.

Has anyone encountered this kind of problem? Could this be due to:

• Incomplete DpnI digestion?
• Antibiotic degradation (allowing satellite colonies)?
• Primer design or amplification issues with large plasmids?
• Something else I'm missing?

Any suggestions or similar experiences would be greatly appreciated!