4. Purify plasmid DNA from 6 to 12 colonies for restriction
analysis and sequencing. We typically first digest 500 ng of
purified plasmid with BamHI/XbaI and XhoI/NheI (both in
CutSmart Buffer) to control the presence of the desired inserts
(see Note 6). In the second step, two positive clones are
selected, and their expression cassettes are sequenced (Table 4).
3.6 Restriction-Free
(RF) Cloning
for the Modification
of the Existing
Constructs
Preparation of multiprotein complexes has its own challenges and
specificities. Typically, constructs might need to be modified by
mutation, truncation, or deletion of low complexity regions and
the position or nature of affinity tags might have to be modified. In
the example shown in Fig. 6, RF cloning is used to introduce a
cleavable affinity tag at the 3
0 extremity of a cDNA in a complex
expression construct.
Restriction-free (RF) cloning, developed for the introduction
of a foreign DNA into a plasmid at any predetermined position, is
based on the overlap extension site-directed mutagenesis and popularized under the name QuickChange mutagenesis (Stratagene™)
[18–20]. The sequence of interest is PCR-amplified using two
hybrid primers designed with complementarity to the desired insert
and the destination plasmid (Fig. 7a). The double-stranded PCR
product is used as a mega-primer for the second amplification
reaction (Fig. 7b). In this step, each of the DNA strands anneals
to the destination vector at the predetermined position and is
extended in a linear amplification reaction that leads to the formation of a double-stranded nicked plasmid. The parental DNA is
then removed by DpnI treatment (Fig. 7c), and the newly synthesized plasmid, containing the DNA insert, is then transformed into
E. coli cells, in which the nicked DNA is ligated by endogenous
enzymatic activity.
1. Collect the plasmid/cDNA required for the first PCR and the
construct that you wish to modify, carefully verify their
sequences, and design the desired construct.
2. Design a pair of hybrid primers with a 5
0 part complementary to
the destination plasmid followed by a 3
0 part complementary to
the insert and compatible melting temperatures (see Note 2).
Oligonucleotides typically have a 18–25 bp overlap (Tm#
50
C) with the destination vector and 18–25 bp overlap
(Tm# 50
C) with the insert of interest.
3. Set up the first PCR (50 μL) to amplify the fragment to be
introduced using high-fidelity polymerase according to the
manufacturer’s instructions (see Note 8). Use 1–50 ng of template DNA containing the sequence of interest if you amplify
your fragment from a plasmid. Use 100–200 ng of template if
you amplify your fragment from cDNA.
Production of Multiprotein Complexes Using the Baculovirus Expression. . .
33
analysis and sequencing. We typically first digest 500 ng of
purified plasmid with BamHI/XbaI and XhoI/NheI (both in
CutSmart Buffer) to control the presence of the desired inserts
(see Note 6). In the second step, two positive clones are
selected, and their expression cassettes are sequenced (Table 4).
3.6 Restriction-Free
(RF) Cloning
for the Modification
of the Existing
Constructs
Preparation of multiprotein complexes has its own challenges and
specificities. Typically, constructs might need to be modified by
mutation, truncation, or deletion of low complexity regions and
the position or nature of affinity tags might have to be modified. In
the example shown in Fig. 6, RF cloning is used to introduce a
cleavable affinity tag at the 3
0 extremity of a cDNA in a complex
expression construct.
Restriction-free (RF) cloning, developed for the introduction
of a foreign DNA into a plasmid at any predetermined position, is
based on the overlap extension site-directed mutagenesis and popularized under the name QuickChange mutagenesis (Stratagene™)
[18–20]. The sequence of interest is PCR-amplified using two
hybrid primers designed with complementarity to the desired insert
and the destination plasmid (Fig. 7a). The double-stranded PCR
product is used as a mega-primer for the second amplification
reaction (Fig. 7b). In this step, each of the DNA strands anneals
to the destination vector at the predetermined position and is
extended in a linear amplification reaction that leads to the formation of a double-stranded nicked plasmid. The parental DNA is
then removed by DpnI treatment (Fig. 7c), and the newly synthesized plasmid, containing the DNA insert, is then transformed into
E. coli cells, in which the nicked DNA is ligated by endogenous
enzymatic activity.
1. Collect the plasmid/cDNA required for the first PCR and the
construct that you wish to modify, carefully verify their
sequences, and design the desired construct.
2. Design a pair of hybrid primers with a 5
0 part complementary to
the destination plasmid followed by a 3
0 part complementary to
the insert and compatible melting temperatures (see Note 2).
Oligonucleotides typically have a 18–25 bp overlap (Tm#
50
C) with the destination vector and 18–25 bp overlap
(Tm# 50
C) with the insert of interest.
3. Set up the first PCR (50 μL) to amplify the fragment to be
introduced using high-fidelity polymerase according to the
manufacturer’s instructions (see Note 8). Use 1–50 ng of template DNA containing the sequence of interest if you amplify
your fragment from a plasmid. Use 100–200 ng of template if
you amplify your fragment from cDNA.
Production of Multiprotein Complexes Using the Baculovirus Expression. . .
33