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3-fragment ligation


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#1 yaniv

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Posted 24 July 2002 - 12:37 AM

hi evry one
did anyone ever did ligation of 3 peaces?
Ive got vector of 5.1kb and 2 inserts of 1.2 eache
can any one tell me what ratio to use?
or if he ever secsead in any 3peace ligation?

#2 sbidw

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Posted 24 July 2002 - 05:02 AM

I have done three way ligations before, but with much smaller fragements (600 - 800 bp). I just did them as normal, but put a real excess of insert in. but be warned: check your restrcition sites for possible formation of concatemers!

#3 yaniv

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Posted 24 July 2002 - 10:44 AM

hi sbidw

thank alot for your advice its good to know that some done it before
i had traid 3 different ratio of insert and vector
1:3:3  1:6:6   1:10:10 and i did ligation for 2 houre in 24 celsius degree and overe night in 16 celsius  degree
ill let you know what i've got
thanks again
yaniv


#4 sbidw

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Posted 24 July 2002 - 10:51 AM

You may find better sucess "cold shocking" your ligations, i.e keeping them at room temp for abit, and then cooling rapidly on ice, then let this warm up slowly to room temp over a few hours, then incubate o/n at 16oC. Alternativly you can do it the other way round by putting them in a polystrene box, filled with room temp water, and leaving them in the cold room overnight to cool down.
good luck, Dave.




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