Biomedical Engineering Reference
In-Depth Information
especially for beginners, not to mention three-fragment ligation.
In addition, the use of cutting sites of restriction enzymes usually
results in the addition of several amino acids at the N- and C-termini
of the target protein, and such addition could cause unexpected
effects on the biochemical properties or expression levels of
proteins [
5
].
Several companies have developed recombinase-based cloning
technologies, including the Invitrogen Gateway cloning technol-
ogy (Chap.
14
)
, Clonetech In-Fusion (Chap.
15
), BioCat Cold-
Fusion, and Red/ET Recombination [
6
-
11
]. However, all the
recombinase-based cloning technologies heavily rely on special kits
containing special vectors, enzymes, or hosts.
Here we present a simple and easy cloning method through
DNA multimers generated by prolonged overlap extension PCR
(POE-PCR) [
12
-
14
]. Different from regular overlap extension
PCR, several modifi cations are made: (a) PCR primers are designed
to be complementary for the amplifi cation of a targeted vector and
an insert gene and (b) DNA multimers are generated by POE-
PCR based on two PCR products featuring the overlap regions at
the 5
ends without the presence of any primers. Such DNA
multimers can be transformed into common laboratory
E
.
coli
hosts (e.g., BL21(DE3), DH5
′
and 3
′
, JM109, TOP10), yielding a chi-
meric plasmid, where
E
.
coli
strains are capable of cleaving assimi-
lated DNA multimers into the circular plasmid. By this new
sequence-independent cloning method, the desired positive col-
ony can be obtained within 1 day without the verifi cation by col-
ony PCR. In our laboratory, we have used this method for
constructing more than 100 plasmids with variable length of inser-
tion fragments from 0.2 to 7.0 kb. Among them, the largest plas-
mid has a size of 14 kb.
α
2
Materials
2.1 Biological and
Chemical Materials
1. New England Biolabs (NEB) Phusion polymerase (Ipswich,
MA). Store at −20 °C (
see
Note 1
).
2. 5× HF PCR buffer or 5× GC PCR buffer for PCR (NEB, pro-
vided with the NEB Phusion polymerase). Store at −20 °C.
3. Deoxynucleotide solution mix (dNTP, 10 mM each). Store at
−20 °C.
4. Oligomers which were synthesized by Integrated DNA
Technologies (San Jose, CA) or other companies. Make the
concentration of oligomers to 100
M by adding appropriate
amount of ultrapure water. Store them at −20 °C till used.
5. Plasmids and/or genomic DNA for PCR amplifi cation template.
6. DNA Clean & Concentrator Kit from Zymo Research (Irvine,
CA) or its equivalent.
μ
