发信人: shakuras (doskey), 信区: Biology
标 题: Re: another clone trick: 3-way ligation
发信站: The unknown SPACE (Fri May 3 18:04:27 2002), 站内信件

I can tell u what I have done in China
Synthesis 10 ssDNA of 60Nts each (indeed, should be 5 dsDNA with
anealing end), ligate 6 together to get Mixture1, ligate the other
4 to get Mixture2, ligate Mixture1, Mixture2 with vector and finally
got the right product. It means to ligate 10 ssDNA (5 ds fragment)
with vector in one step, and it works!!!!
【 在 whatwhyhow (幽克拉) 的大作中提到: 】
: nice trick. :)
: but i did that 5 years ago :) people in our lab did that all the time, though
: i didn't find it very efficient :( maybe my bench technique was not as perfect
: as yours. :)
: 【 在 leohawk (leohawk) 的大作中提到: 】
: : Well, I am sure I am not the first to do this, but I do want to share it
: : Sometimes, cloning multiple things into a vector can be tedious as you
: : have to transfer the fragments through several vector to get the
: : desired one, usually because the existence of the same Restriction site
: : in the vector and the fragment you want to clone.
: : say a gene has 2 frags in two separate vector, now you wanna get
: : them ligated into the same vector.
: : EcoRI------------XhoI Frag I (has interal BamHI site)
: : XhoI-------------BamHI Frag II(has internal EcoRI site).
: : just cut a vector with EcoRI/BamHI and put all three fragment in the
: : ligation system...
: : you know what, it works with amazing efficiency! My boss was very amazed
: : when he see it worked so efficiently.
: : never tried 4-way clone, but it might worth a try....
: : hehe...


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