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Has anyone else encounted problems in cloning with PET-22b vector? - the plasmid even smaller than the empty PET-22b vector! (Dec/22/2006 )

Dear all:
I feel extremely frustrated these days because I failed several times in a seemly simple experiment: to insert a cDNA fragment into the PET-22b vector (Novagene)! The PET-22b vector and the PCR amplified cDNA were both double digested by SalI and NdeI and then they were ligated. What is strange is that after transformed into the E.Coli JM109 line, the clones emerged always contained plasmid even smaller than the empty PET-22b vector!
Please give me a hand if you know why, thanks very much!

-peixumol-

QUOTE (peixumol @ Dec 22 2006, 08:52 PM)
Dear all:
I feel extremely frustrated these days because I failed several times in a seemly simple experiment: to insert a cDNA fragment into the PET-22b vector (Novagene)! The PET-22b vector and the PCR amplified cDNA were both double digested by SalI and NdeI and then they were ligated. What is strange is that after transformed into the E.Coli JM109 line, the clones emerged always contained plasmid even smaller than the empty PET-22b vector!
Please give me a hand if you know why, thanks very much!


I dont know the reason for your problem, but i think you can try another pET22b clone.

-subberry-