i failed to clone a 2.2 kb gene in to a vector (about 12 kb). anybody can give me some oppinion and suggestion? below are the details
TAKARA pRI201-AN. cut with SacI, treated with T4 DNA polimerase to produce blunt ends, the cut with NdeI to product a linear vector with sticky end at one side and blunt end at another side. gel purified
PCR product amplified with Pfu dna polymerase ( blunt end), cut with NdeI at to produce stiky end at one side. ge purified
90ng of vector and about 47ng of insert. (vector: insert =1:3)
in 20ul reaction
all of the colonies i obtained are with out insert, seem like the vector ligate with another vector ( sticky end bind with sticky end, blunt end with blunt)
I really hpe somebody can help me to solve this problem.
Thank you very much.