ligation trouble - (Apr/09/2006 )

I have troubles when ligate a 2.3kb insert into a 10 kb vector. Insert was cutted by xbaI and salI and vector by NheI and xhoI. I can not get recombinat clone several times?
anyone do the similar work? thank you

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Why do you use 4 different enzymes. If you want to promote Unidirectional cloning, just use 2 enzymes that are on your vector as are on your insert. Neither of your enzymes are isoschizomers of the other.
Ensure that the enzyme you pick ensure directional cloning and they do not cut anywhere on your insert.

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To perhaps help you,

http://www.neb.com/nebecomm/tech_reference...hizomers.asp?#X

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