Hi there, I used to digest pET28 vector and my insert sized 700bp with BamHI and HindIII prior to ligation. The insert was amplified by PCR using specific primers with the respective RE. However, sequence result showed that the target gene was inserted to the vector at location of HindIII only (the insert carries ~ 100bp of TOPO sequence). I wonder why the BamHI is not working? I use BamHI buffer and all enzymes are from Fermentas. Many thanks
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