Hello, did you try vector treatment with phosphatase (SAP, CIP etc) with subsequent purification? What kind of ends do your restriction produces? Blunt? Sticky? If sticky, then cohesive or not?

There is not enough information in your question to give good answer, so I can only suppose most probable pitfall.

@Anatoliy Zubritskiy, I haven't treat with phophatases. Restriction produces Sticky, cohesive endings. May be I have to treat with phosphatases.

Muluken Kebede Yes, in this case you'd treat your vector with phosphatase after restriction to avoid self-ligation. Good luck!

@Anatoliy Zubritskiy, Many thanks!

https://www.addgene.org/protocols/plko/

I follow the protocol and it totally works for me. Make sure that Age1 and EcorI work well and importantly, your DH5alpha must be good.

Thank you very much. I use Stbl3 strain.

Stbl3 is more difficult to transform than DH5alpha. You can try DH5alpha for cloning and then use Stbl3 for producing DNA. Hope it helps.