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ligate 3 DNA fragments with sticky ends into Vector


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#1 uioli

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Posted 15 June 2002 - 03:54 PM

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,



#2 milanoj

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Posted 20 June 2002 - 12:24 PM

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.




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