Hello there,

I'm having some issues with digesting a PCR product and using it for ligation with the vector pET-28a.

My PCR product is about ~1200 bp, I purify it and get about 30 ng/uL. Then I digest it using NotI and NcoI enzymes, but when I run the digestion what I get are two bands, one about 1200bp and another about 1000bp (the digestion should result in a ~800 bp product). The digestion using only NcoI or only NotI should get a ~900 bp fragment. I even tried to use that ~1000 bp with the digested pet-28a vector in ligation to see what I would get, but I get no colony (I'm using BL21(DE3)) at all.

Any suggestions?

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