ligate 3 DNA fragments with sticky ends into Vector-Mole

 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.

-uioli-

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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.

-milanoj-

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