Amplifying a plasmid backbone can sometimes be challenging, especially when non-specific priming occurs. Here are some steps you can take to increase the specificity of your PCR:

By following these steps, you should be able to optimize your PCR conditions and increase the specificity of your plasmid backbone amplification.@Vanessa Sewell

can add DMSO（Dimethyl sulfoxide ） in your PCR recation mix

In addition to the excellent suggestions above, here are my thoughts:

If you are trying to amplify a large backbone, say over 3kbp, perhaps it is better to excise the unwanted DNA with restriction enzymes and ligate the plasmid back with the gene? That way, bacteria (e.g. E. coli DH5α) can simply produce the new plasmid in large quantities.

Also, Q5 may not have the processivity required to accurately amplify your backbone, even if the primers are specific and well designed. The phusion taq polymerase has very good processivity, and may be more suitable. Make sure that you have enough dNTPs and magnesium in the reaction, otherwise the polymerase may insert the wrong nucleotide.

Generally speaking, inserting the same gene several times into a plasmid is inefficient. It is usually easier to choose a high copy number plasmid coupled with a strong promoter, rather than several copies of a given gene. This also saves you the headache of finding out the exact number of copies.

Amplifying a plasmid backbone without non-specific priming can be challenging due to the high homology between plasmid backbones and the presence of repetitive elements. However, here are some tips that can help to minimize non-specific priming and improve the specificity of your PCR reaction:

This video playlist might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE