2. Decide on the constructs to be generated and select the transfer
vector accordingly. Consider, for example, the presence and
position of affinity tags.
3. Design a pair of generic primers to amplify the vector backbone
(Backbone-F and Backbone-R) and another one for the dual
promoter module (Prom-F and Prom-R). Whenever possible,
oligonucleotides Prom-F and Prom-R contain a kozak consensus sequence (CCACCATGG, where ATG encodes the initiator Methionine) and are perfectly complementary to their
vector template.
4. Design pairs of specific primers to amplify the target cDNAs
(pH-gene-START-F, pH-gene-STOP-R, p10-gene-START-F
and p10-gene-STOP-R). They should be composed of a 5
0
part corresponding to the cloning junction (which provides a
homology to the vector sequence for the homology-based
cloning reaction) followed by a 3
0 part complementary to the
insert target (which provides a priming region for the PCR).
The START primers include the first 15–21 nt of the cDNA,
preceded by a sequence complementary to that of the oligonucleotide used to amplify the promoter region. The STOP primers include the last 15–21 nt of the gene, followed by a STOP
codon and the reverse complement of the primers used for the
backbone amplification.
5. Simulate the assembly of the expected transfer plasmids in silico
and carefully check that the tags, if any, are inserted in phase
with the genes of interest.
6. Check the propensity of the primers to form secondary structures or dimerize. Structures with a ΔG greater than À6 kcal/
mole suggest that redesign may be required, particularly for
those at the 3
0 end, for example, by increasing the length of the
5
0 -tail.
7. Order the oligonucleotides.
By default, oligonucleotides are chosen to generate PCR fragments with 20 nucleotide-overlapping ends. They are usually
designed taking into account the length and base composition of
each primer pair, so that the Tm of the primers/templates are well
matched and close to 60
C (see Note 2). To minimize the formation of secondary structures, instead of using long oligonucleotides
(e.g., to append an affinity or detection tag), do not hesitate to use
nested PCRs.
ä
Fig. 4 (continued) Prom-R/Prom-F pair. The eGFP and mCherry cDNAs are amplified with the p10-geneSTART-F/p10-gene-STOP-R and the pH-gene-START-F/pH-gene-STOP-R. Note that oligonucleotides for the
amplification of the “promoter” cassette and of the 5
0
end of the cDNAs are designed to contain a Kozak
consensus CCACCATGG sequence where ATG (in bold) encodes the initiator codon (see Table 2)
Production of Multiprotein Complexes Using the Baculovirus Expression. . .
27
vector accordingly. Consider, for example, the presence and
position of affinity tags.
3. Design a pair of generic primers to amplify the vector backbone
(Backbone-F and Backbone-R) and another one for the dual
promoter module (Prom-F and Prom-R). Whenever possible,
oligonucleotides Prom-F and Prom-R contain a kozak consensus sequence (CCACCATGG, where ATG encodes the initiator Methionine) and are perfectly complementary to their
vector template.
4. Design pairs of specific primers to amplify the target cDNAs
(pH-gene-START-F, pH-gene-STOP-R, p10-gene-START-F
and p10-gene-STOP-R). They should be composed of a 5
0
part corresponding to the cloning junction (which provides a
homology to the vector sequence for the homology-based
cloning reaction) followed by a 3
0 part complementary to the
insert target (which provides a priming region for the PCR).
The START primers include the first 15–21 nt of the cDNA,
preceded by a sequence complementary to that of the oligonucleotide used to amplify the promoter region. The STOP primers include the last 15–21 nt of the gene, followed by a STOP
codon and the reverse complement of the primers used for the
backbone amplification.
5. Simulate the assembly of the expected transfer plasmids in silico
and carefully check that the tags, if any, are inserted in phase
with the genes of interest.
6. Check the propensity of the primers to form secondary structures or dimerize. Structures with a ΔG greater than À6 kcal/
mole suggest that redesign may be required, particularly for
those at the 3
0 end, for example, by increasing the length of the
5
0 -tail.
7. Order the oligonucleotides.
By default, oligonucleotides are chosen to generate PCR fragments with 20 nucleotide-overlapping ends. They are usually
designed taking into account the length and base composition of
each primer pair, so that the Tm of the primers/templates are well
matched and close to 60
C (see Note 2). To minimize the formation of secondary structures, instead of using long oligonucleotides
(e.g., to append an affinity or detection tag), do not hesitate to use
nested PCRs.
ä
Fig. 4 (continued) Prom-R/Prom-F pair. The eGFP and mCherry cDNAs are amplified with the p10-geneSTART-F/p10-gene-STOP-R and the pH-gene-START-F/pH-gene-STOP-R. Note that oligonucleotides for the
amplification of the “promoter” cassette and of the 5
0
end of the cDNAs are designed to contain a Kozak
consensus CCACCATGG sequence where ATG (in bold) encodes the initiator codon (see Table 2)
Production of Multiprotein Complexes Using the Baculovirus Expression. . .
27