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.
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.
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.
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.
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: