Biomedical Engineering Reference
In-Depth Information
of both PCR products will result in a construct lacking BpiI
restriction sites. Restriction and ligation can therefore be per-
formed at the same time since the ligated product will not be
redigested with BpiI. Restriction-ligation is more effi cient than
restriction followed by ligation, and will lead to a high propor-
tion of correct constructs [
13
].
4. Using type IIS restriction enzymes and restriction-ligation will
lead to a high cloning effi ciency. However, one possible type of
non-correct construct that could be obtained consists of
pUC19 vector that may be carried over from the template used
for PCR amplifi cation in Subheading
3.1
,
steps 2
and
3
. This
will produce blue colonies, as with the desired QC cloning vec-
tor. One solution to avoid these clones is to amplify a
lacZ
fragment from a vector carrying a different antibiotic marker
(for Subheading
3.1
,
step 2
), and to amplify the backbone
fragment from a pUC19-derived plasmid already containing
an insert, and therefore with a nonfunctional
lacZ
α
α
gene (for
Subheading
3.1
,
step 3
).
5. It is useful to sequence the vector in the region corresponding
to the adaptor and the catching sequences, since both could
contain mutations derived from the primer. Sequencing the
rest of the plasmid is not important since the plasmid is repli-
cating and is therefore functional, and the
lacZ
fragment pro-
vides a blue color and is therefore also functional.
6. Inactivation of PstI is necessary, as incubation of the digested
vector with insert (in subsequent Subheading
3.3
,
step 2
)
might lead to digestion of inserts that would contain a PstI site
in the unknown region. Such digestion would of course pre-
vent cloning of these inserts. PstI digestion of the vector and
the following heat inactivation step could be performed in a
water bath, but are more conveniently performed in a thermal
cycler.
7. Even though the amount of DNA has been quantifi ed using a
NanoDrop device or any other method, it is always a good idea
to confi rm visually the amount and the quality of purifi ed PCR
product by agarose gel electrophoresis.
8. We use T4 DNA ligase buffer, but since the reaction does not
require ATP, other buffers can be used instead; e.g., NEBuffer
2 is suitable for QC cloning.
9. The quality of T4 DNA polymerase is critical. It is important
to check that the enzyme lot for the T4 DNA polymerase is
not expired as cloning might become less effi cient. If cloning
has failed several times for unknown reasons, one should try
another lot of enzyme from the same manufacturer, or enzyme
from another manufacturer. In practice, we obtained good
results using the T4 DNA polymerase supplied by Fermentas
(cat. no. EP0061).
α
