2.4 Reagents
for a SLIC Reaction
1. T4 DNA Polymerase (New England Biolabs).
2. 10Â NEB 2.1 buffer (Tris-HCl 100 mM, MgCl 2 100 mM,
NaCl 500 mM, BSA 1 mg/mL, pH 7.9).
3. 10 mM dCTP.
4. 10Â Ligation buffer (e.g., Tris-HCl 500 mM, MgCl 2 100 mM,
DDT 100 mM, pH 7.5) or any DNA ligase buffer for
annealing.
5. 1 μg of linearized vector and of each amplified DNA fragment
to be assembled prepared as described in Subheading 3.3.
2.5 Reagents for an
In-Fusion Reaction
1. In-Fusion or Gibson assembly kit.
2. 1 μg of linearized vector and of each amplified DNA fragment
to be assembled prepared as described in Subheading 3.3.
2.6 Solution
and Media
for Restriction-Free
Cloning
1. Phusion Polymerase (ThermoScientific™) with 5xHF or GC
buffer and DMSO or another high-fidelity polymerase.
2. dNTPs mix. Prepare and aliquot 10 mM dNTP stock solutions
in ultrapure water.
DpnI (10 u/μL) restriction enzyme and corresponding
buffer.
3 Methods
Here we detail the preparation of dual expression plasmids using
homology-based cloning and the modification of existing constructs. We focus on an expression cassette composed of two divergent baculovirus late promoters (PH and p10), each followed by a
multiple cloning site (MSC1 and MSC2) and a transcription termination signal (SV40 pA and HSV-TK pA) (Fig. 2a). This cassette
organization is found in the transfer vector FastBac Dual and in its
Multibac derivatives which include the acceptor vectors pFL and
pKL and the donor plasmids pUCDM and pSPL (Table 1). It was
also inserted into the backbone of the transfer vector pBacPack8
(plasmid pAC8_MF) to enable the generation of recombinant
baculoviruses by homologous recombination directly in insect
cells (Fig. 2b). In a typical experiment, the plasmid backbone is
combined with the promoter module (comprising the PH and p10
promoters) and cDNAs encoding the two target genes, in a fourfragment assembly reaction (Fig. 3). The first cDNA is typically
inserted between the BamHI and XbaI sites of MCS1 and the
second between the XhoI and NheI sites of MSC2 (Fig. 2a).
Following a general outline of homology-based technologies
for fragment assembly in Subheading 3.1, we detail primer selection in Subheading 3.2 and provide protocols for the preparation
Production of Multiprotein Complexes Using the Baculovirus Expression. . .
23
for a SLIC Reaction
1. T4 DNA Polymerase (New England Biolabs).
2. 10Â NEB 2.1 buffer (Tris-HCl 100 mM, MgCl 2 100 mM,
NaCl 500 mM, BSA 1 mg/mL, pH 7.9).
3. 10 mM dCTP.
4. 10Â Ligation buffer (e.g., Tris-HCl 500 mM, MgCl 2 100 mM,
DDT 100 mM, pH 7.5) or any DNA ligase buffer for
annealing.
5. 1 μg of linearized vector and of each amplified DNA fragment
to be assembled prepared as described in Subheading 3.3.
2.5 Reagents for an
In-Fusion Reaction
1. In-Fusion or Gibson assembly kit.
2. 1 μg of linearized vector and of each amplified DNA fragment
to be assembled prepared as described in Subheading 3.3.
2.6 Solution
and Media
for Restriction-Free
Cloning
1. Phusion Polymerase (ThermoScientific™) with 5xHF or GC
buffer and DMSO or another high-fidelity polymerase.
2. dNTPs mix. Prepare and aliquot 10 mM dNTP stock solutions
in ultrapure water.
DpnI (10 u/μL) restriction enzyme and corresponding
buffer.
3 Methods
Here we detail the preparation of dual expression plasmids using
homology-based cloning and the modification of existing constructs. We focus on an expression cassette composed of two divergent baculovirus late promoters (PH and p10), each followed by a
multiple cloning site (MSC1 and MSC2) and a transcription termination signal (SV40 pA and HSV-TK pA) (Fig. 2a). This cassette
organization is found in the transfer vector FastBac Dual and in its
Multibac derivatives which include the acceptor vectors pFL and
pKL and the donor plasmids pUCDM and pSPL (Table 1). It was
also inserted into the backbone of the transfer vector pBacPack8
(plasmid pAC8_MF) to enable the generation of recombinant
baculoviruses by homologous recombination directly in insect
cells (Fig. 2b). In a typical experiment, the plasmid backbone is
combined with the promoter module (comprising the PH and p10
promoters) and cDNAs encoding the two target genes, in a fourfragment assembly reaction (Fig. 3). The first cDNA is typically
inserted between the BamHI and XbaI sites of MCS1 and the
second between the XhoI and NheI sites of MSC2 (Fig. 2a).
Following a general outline of homology-based technologies
for fragment assembly in Subheading 3.1, we detail primer selection in Subheading 3.2 and provide protocols for the preparation
Production of Multiprotein Complexes Using the Baculovirus Expression. . .
23