Is this clone the correct one? - Restriction digestion doubt (Aug/19/2007 )
Can someone help me with the followng cloning problem:
I digested plasmid DNA with 2 restriction enzymes ApaI (GGGCC/C) and XbaI ( T/CTAGA)
after I used Klenow to fill in and ligated with T4 DNA ligase.
I expected to get as a results the following DNA sequence: TCTAGGGCCC
but instead I got the following : TCTAGC
Is the clone I got, correct?
yes, the clone you have is correct.
The ApaI restriction enzyme leave a 3' over hang. Klenow have a 3'-5' exonuclease activity. So rather then filling in a 3' overhang, it will remove said overhang, leaving a blunt end