subclone a double-cut insert - (Aug/18/2006 )

hi I would like to clone a fragment generated by a double cute EcoRI and Kpn1 in a plasmid dublecuted with Xba1/sal1 or single cuted with Sal1 or Xba1.

any advice
anyprotocole
thanks

ulujm

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either you PCR your insert with primers containing the right recognition sequence for the restriction enzymes you will use tu cut the plasmid, or you use a kleenow enzyme that will transform the cohesive ends into blunt ends (which will be compatible then), and then you can ligate the blunt ends.

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thanks but if I cut with EcoR1/KPN1 I got:
5' AATTC---------------GGTAC 3'
3' ____G--------------C ____5'

the G should be below the C of AATTC and the C below the G of GGTAC

will the klenow fill the 5'hanging and 3' hanging in the same time.
what type of klenow do I need.


thanks

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Sorry, I think I was wrong with KpnI, because Klenow is a 5'-3' polymerase, and will not feel the KpnI end, if I'm right.

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if I check on biolab catalogue page 87 there is DNA polI large klenow fragment.

-in the application I can read fill in of 5' to form blunt ends
-removal of 3' overhangs to form blunt ends

so I guess I can order this:

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