2 Materials
2.1 Working
Environment
and Instruments
You need access to standard equipment for molecular biology
including PCR machines, agarose gel electrophoresis apparatus,
UV trans-illuminator, refrigerated benchtop centrifuge, nanodrop
for DNA quantification, fridge, and freezers (À20
C and À80
C).
Prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a sensitivity of 18 MΩ-cm at 25
C)
and analytical grade reagents. Unless indicated otherwise, prepare
and store all reagents at room temperature. Diligently follow all
waste disposal regulations when disposing waste materials.
2.2 Bacterial Strains
and Plasmids
1. Luria–Bertani (LB) medium with antibiotics added at the following concentrations: ampicillin (100 μg/mL), kanamycin
(30 μg/mL), spectinomycin (90 μg/mL), and chloramphenicol (15 μg/mL).
2. E. coli DH5α, TOP10, XL-1 blue, or Pir1 (see Note 1) competent cells (chemically or electrocompetent cells).
3. Dual expression transfer vectors (see Table 1). These plasmids
are available from Addgene.
4. Templates for amplification of the target genes. When a starting
plasmid containing the cDNA of your gene of interest is not
present, chemical synthesis is often an advantageous option for
generating cDNAs. In any case, the removal of restriction sites
that might be used for the transfer of the expression cassettes is
Table 1
Dual promoter plasmids for baculovirus expression systems
Plasmid
Method
Resistance
Size
Origin
References
pFastBac Dual Tn7 transposition
Ampicillin,
gentamicin
5.2 kbp pUC ori None
pFL
Tn7 transposition
Ampicillin,
gentamicin
5.8 kbp ColE1
[6]
pKL
Tn7 transposition
Kanamycin,
gentamicin
4.9 kbp pBR322
[5]
pAC8_MF
Homology
recombination
Ampicillin
6.9 kbp pUC ori Kolesnikova
et al. (in prep)
pUCDM
a
Cre-mediated fusion Chloramphenicol 3.0 kbp R6Kγ
[5]
pSPL
a
Cre-mediated fusion Spectinomycin
2.9 kbp R6Kγ
[6]
These plasmids contain the expression cassette detailed in Fig. 2a and with the exception of pFastBac Dual all possess a
LoxP site. Vectors pFastBac Dual, pFL, and pKL use Tn7 transposition while pAC8_MF relies on homology recombination to integrate the expression cassette in the viral DNA
a
Vectors pUCDM and pSPL cannot be used directly to generate recombinant viruses but can be fused to pFL, pKL, and
pAC8_MF through using Cre-mediated reactions
20
Paola Rossolillo et al.
2.1 Working
Environment
and Instruments
You need access to standard equipment for molecular biology
including PCR machines, agarose gel electrophoresis apparatus,
UV trans-illuminator, refrigerated benchtop centrifuge, nanodrop
for DNA quantification, fridge, and freezers (À20
C and À80
C).
Prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a sensitivity of 18 MΩ-cm at 25
C)
and analytical grade reagents. Unless indicated otherwise, prepare
and store all reagents at room temperature. Diligently follow all
waste disposal regulations when disposing waste materials.
2.2 Bacterial Strains
and Plasmids
1. Luria–Bertani (LB) medium with antibiotics added at the following concentrations: ampicillin (100 μg/mL), kanamycin
(30 μg/mL), spectinomycin (90 μg/mL), and chloramphenicol (15 μg/mL).
2. E. coli DH5α, TOP10, XL-1 blue, or Pir1 (see Note 1) competent cells (chemically or electrocompetent cells).
3. Dual expression transfer vectors (see Table 1). These plasmids
are available from Addgene.
4. Templates for amplification of the target genes. When a starting
plasmid containing the cDNA of your gene of interest is not
present, chemical synthesis is often an advantageous option for
generating cDNAs. In any case, the removal of restriction sites
that might be used for the transfer of the expression cassettes is
Table 1
Dual promoter plasmids for baculovirus expression systems
Plasmid
Method
Resistance
Size
Origin
References
pFastBac Dual Tn7 transposition
Ampicillin,
gentamicin
5.2 kbp pUC ori None
pFL
Tn7 transposition
Ampicillin,
gentamicin
5.8 kbp ColE1
[6]
pKL
Tn7 transposition
Kanamycin,
gentamicin
4.9 kbp pBR322
[5]
pAC8_MF
Homology
recombination
Ampicillin
6.9 kbp pUC ori Kolesnikova
et al. (in prep)
pUCDM
a
Cre-mediated fusion Chloramphenicol 3.0 kbp R6Kγ
[5]
pSPL
a
Cre-mediated fusion Spectinomycin
2.9 kbp R6Kγ
[6]
These plasmids contain the expression cassette detailed in Fig. 2a and with the exception of pFastBac Dual all possess a
LoxP site. Vectors pFastBac Dual, pFL, and pKL use Tn7 transposition while pAC8_MF relies on homology recombination to integrate the expression cassette in the viral DNA
a
Vectors pUCDM and pSPL cannot be used directly to generate recombinant viruses but can be fused to pFL, pKL, and
pAC8_MF through using Cre-mediated reactions
20
Paola Rossolillo et al.