Biomedical Engineering Reference
In-Depth Information
nonspecifi c hybridization and leads to compromised cloning
effi ciency and accuracy, CPEC designs the overlapping ends to
have very similar
Tm
(±2 °C) and performs the annealing step at
high, stringent temperatures (typically in the range of 55-65 °C)
to ensure highest accuracy in multi-way assembly and complex
library cloning. Unlike PCR, CPEC does not amplify sequences
and therefore does not propagate errors with an increased number
of thermal cycles.
The combinatorial library cloning strategy using CPEC is
illustrated in Fig.
2
. In this example, two libraries are cloned simul-
taneously into a single vector for expression or functional screens
to identify the best combinatorial sequences. It is anticipated that
such screens will be performed more and more frequently in syn-
thetic biology applications to construct and identify the optimal
macromolecular complexes or gene networks. So far, CPEC is the
only in vitro method that works well in our hands for combinato-
rial library cloning [
5
]. The successful development of this cloning
strategy will highly accelerate the process of protein expression
screening using large quantity of library gene variants and hence
the development of synthetic biology applications as such screens
will be performed more and more frequently to construct and
identify the optimal macromolecular complexes or gene networks.
2
Materials
2.1 Reagents
1. Linearized cloning vector: Commercial or custom-designed
vectors can be used.
2. Cloning insert or insert library: Cloning insert can be prepared
by PCR or restriction digestion from a particular plasmid or
DNA template. Insert library is often assembled using oligo
libraries synthesized with a DNA synthesizer in house or from
commercial providers such as IDT.
3. dNTP mix (dATP, dCTP, dGTP, and dTTP) (e.g., Bioline, cat.
no. BIO-39043).
4. Oligonucleotide primers: Custom DNA primer synthesis is
available from commercial suppliers such as IDT. Prepare stock
solutions of primers (e.g., 100
M) using sterile DNase/
RNase-free water. Prepare aliquots of 10× working solution
(e.g., 10
μ
M) and store at −20 °C to prevent contamination of
stock and repeat freeze-thaw cycles.
5. Phusion High-Fidelity DNA polymerase with 5× Phusion HF
buffer (Finnzymes, cat. no. F-530).
6. Nuclease-free water (Sigma-Aldrich, cat. no. W4502).
7. Taq DNA polymerase with ThermoPol buffer (New England
Biolabs, cat. no. M0267).
μ
