ligate 3 DNA fragments with sticky ends into Vector - (Jun/15/2002 )
Hi, Dear All,
Have you ever tried to ligate 3 DNA fragments with sticky ends into Vector(5.2kb).
A. Fragment: 3.0kb, with HindIII/ Bam HI cut from a Vector.
B. Fragment: 0.5kb, BamHI / XbaI cut from a Vector.
C. Fragment: 3.5kb, XbaI/ NotI cut from a Vector.
firstly, I cut A, B out of vector, then ligate them, run a gel, cut the right size band(A+B=3.5kb), recover it, then Conform the DNA by BamHI cut.
But it is really a tiny amount of DNA each time.
then I want to put A+B into a Vector. If I tried 3:1 ratio(insert: Vector), I never have enough DNA. So I failed several times with my trasformation.
Are there any better ways to solve this problem?
Thanks a lot,
I think to save yourself much frustration you can perform overlap/extension PCR to fuse your fragments. See Methods in Molecular Biology Vol. 57 In vitro mutagenesis Protocols. Alternatively you can clone one fragment then the other.