cloning problem - (Mar/31/2010 )

For how long you incubate your ligation reaction? If I were you I would do the ligation at 4 degrees overnight. When I have an insert larger than 2kb, I always use these conditions.

Check your ligation by ligating a.) CIP treated and b.) untreated cut vector. So you can test if your ligation works and if your CIP treatment is OK. BTW I treat my vector with CIP usually just for one hour, it's more then enough for my protocol.