Hi,

I am trying to amplify a 9856 bp segment from a 10012 bp plasmid by PCR in order to use it for Gibson assembly.

When I run the gel, one of my other fragments that I am trying to amplify by PCR works very nicely, giving me a single band (lane 3), whilst the 9856 bp segment looks quite dirty and it is also giving be an extra 500bp band (lane 5).

How can I improve the PCR?

I have attached the details of the PCR, thermocycling and the gel (0.8% agarose, 1kb GelRuler ladder from ThermoFisher).

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