The PCR product has to be digested with the same restriction endonuclease as
the vector in order to create cohesive ends, as the ligation of blunt-ended fragments
yields poor results (K 
for the activity of T4 ligase on blunt-ended
DNA is nearly 100 times higher than its K on DNA with cohesive
ends).
Furthermore, in contrast to blunt end cloning, it is not relevant in
overhanging end cloning whether or not terminal dAs are added to the PCR product, since
they are removed if present.
5 
l of the ethanol precipitated PCR product (see section PCR) are
digested with 20 U (1 l) EcoRI
(New England Biolabs) in 20 l
volume for 90 minutes at 37psy176 C. The digest is separated on agarose, and
the insert band cut out. After eluating the DNA (see section freeze),
a phenol extraction (see section phenol) and ethanol precipitation (see
section 2.5.5) followed. The final DNA pellet was resuspended in 10
l water and now ready for ligation.