My first ever 3-way ligation :) - Advice on ratios? (Sep/25/2008 )

Hey everyone

I am just about to attempt my first ever 3-way ligation. I have never had problems in the past with 2-way but just wanted to ask your advice first...

Do I use the same kind of ratios as I would for a 2-way?

I have a 5.4kb vector and two inserts (2.4kb and 1.7kb) and it goes a lil' something like this:

----Not1--Insert 2.4kb
--EcoRV--Insert 1.7kb
--Not1--Vector 5.4kb----

Any advice would be greatly appreciated
Clare

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You still need equal numbers of ends with a three-way ligation.

EcoRV--Insert 1.7kb: 11 ng = 10 fmoles = 1.2 e10 ends
Not1--Insert 2.4kb: 15.6 ng = 10 fmoles = 1.2 e10 ends
Not1--Vector 5.4kb: 35 ng = 10 fmoles = 1.2 e10 ends

Good luck.

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Ahh thanks I'll give it a go next week and let you know how I go
Clare

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Yes, you do need a 1:1:1 molar ratio between your DNA fragments and vector.

also try to avoid using blunt end in a multiway ligations. It adds an extra bit of difficulty.

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Thanks Alas, I have to use EcoRV - there is no other way!!!


Oh, I just realised I have never worked with blunt ends before either! I think I've been a lucky cloner...until now

Clare

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I hope your luck continues!

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