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help: three fragment ligation - (Jul/25/2005 )

I am currently working on three fragment ligation. the vector is 5.6kb, one insert is 7.3kb, the second one is 6.2 kb. none of them was generated with blunt restriction enzyme. no oritation issue, did do dephyspholation with SAP. I got colonines but with single insert(sounds weird). my questions are:

1 cannt the vector stand that large insert?
2 how can I check the effect of de-P except control self ligation?
3 in case it is vector issue, any recommedation of vector that can hold 20kb insert?

looking forward your input




A cosmid or fosmid would be a good choice for long inserts. Check pBelobac11 or pFos1 plasmids.