Hello,

I am trying to ligate a fragment of 4.7 kb into pENTR4 dual selection vector with one restriction enzyme only. I performed the digestion reaction with NotI, then I purified the corresponding bands and dephosphorilate pENTR4 with CIP, ligate the fragment with the vector using T4 DNA ligase for 1 h, then I transformed with NEB® 5-alpha Competent E. coli (High Efficiency) but I cannot obtain any colony, any suggestion?

thanks