I did an overlap-extension PCR and got the following gel image (purple image +
neb 1k ladder). The target band size is 1.9kb, which was the brightest band.
Afterwrds, I cut the 1.9kb bands and gel purified and PCR-ed using same set of primers, and I got exactly the same non-specific band patterns (black & white image).
I used Phusion & HF buffer. I used this protocol for OE-PCR. https://www.protocols.io/view/overlap-extension-pcr-x54v9xkzv3eq/v1
May I ask why does gel purification does not completely get rid of non-specific bands from OE-PCR, and how should I get rid of these bands for subsequent cloning?
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