Hi Lifang,

Please elaborate the question so others can understand the process you've been through. To start troubleshooting, what's the length of insert and vector? when the ligated DNA was transformed into E. coli, have you used vector or insert along as control? This can test out whether the ligation was successful, by comparing the colony number. For the PCR part, I'm not quite understand if it was performed before ligation (that you performed first PCR, then ligate into expression vector), or have you performed after transformation ( to test if DNA was ligated correctly)?

Thank you for your answer, the problem was solved when I replaced the new carrier.@Mu-Shen Chang