For Research Use Only. Not for use in diagnostic procedures.
Bac-to-Bac™ Baculovirus Expression System
USER GUIDE
An efficient site-specific transposition system to generate
baculovirus for high-level expression of recombinant proteins
Catalog Numbers 10359-016, 10360-014, 10584-027, 10712-024
Publication Number MAN0000414
Revision B.0
Manufacturer: Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, CA 92008
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,
INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,
INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0000414
Revision Date Description
B.0 16 July 2018 Rebrand
A.0 17 August 2015 Baseline for revisions
Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the
terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
©2018 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■Product information ....................................................... 8
Product description ............................................................. 8
Kit contents and storage ......................................................... 8
Types of products ........................................................... 8
Shipping and storage ........................................................ 8
pFastBac™ vectors .......................................................... 9
MAX Efficiency™ DH10Bac™ Competent
E. coli
.................................. 9
ExpiFectamine™ Sf Transfection Reagent ..................................... 10
The Bac-to-Bac™ Baculovirus Expression System .................................. 10
System components ........................................................ 10
pFastBac™ vector .......................................................... 10
DH10Bac™
E. coli
.......................................................... 10
Baculovirus shuttle vector .................................................. 11
Helper plasmid ............................................................ 11
ExpiFectamine™ Sf Transfection Reagent ..................................... 11
Diagram of the Bac-to-Bac™ System ......................................... 12
Workflow ................................................................. 13
■Methods ................................................................... 14
Culture insect cells ............................................................. 14
Introduction ............................................................... 14
Serum-free medium ....................................................... 14
Insect cell culture reference guide ........................................... 14
General cell culture guidelines .............................................. 15
Cells for transfection ....................................................... 15
Generate recombinant pFastBac™ donor plasmid .................................. 15
Introduction ............................................................... 15
Select
E. coli
host .......................................................... 15
Transformation method .................................................... 16
Analyze the transformants .................................................. 16
Analyze the transformants by PCR ........................................... 16
Bac-to-Bac
™
Baculovirus Expression System User Guide
3
Sequencing ............................................................... 16
Make a glycerol stock for long-term storage .................................. 16
Generate recombinant bacmid DNA: transform DH10Bac™
E. coli
.................... 17
Introduction ............................................................... 17
Positive control ............................................................ 17
Required materials ........................................................ 17
Recommendation .......................................................... 18
Prepare for transformation ................................................. 18
Transform DH10Bac™
E. coli
................................................ 18
Verify the phenotype ....................................................... 19
Isolate recombinant bacmid DNA ................................................. 20
Introduction ............................................................... 20
Before you begin ........................................................... 20
Equilibrate the column ..................................................... 20
Prepare the cell lysate ...................................................... 21
Bind and wash the DNA ..................................................... 21
Elute and precipitate DNA .................................................. 21
Analyze recombinant bacmid DNA by PCR ......................................... 22
Introduction ............................................................... 22
Analyze by PCR with pUC/M13 primers ....................................... 23
DNA polymerase ........................................................... 23
Generate the PCR product .................................................. 23
What you should see ....................................................... 24
Produce recombinant baculovirus: transfect insect cells ............................ 25
Introduction ............................................................... 25
Plasmid preparation ....................................................... 25
ExpiFectamine™ Sf Transfection Reagent ..................................... 25
Insect cell lines ............................................................ 25
Media for transfection ...................................................... 25
Positive control ............................................................ 25
Required materials ........................................................ 25
Transfection conditions ..................................................... 26
Important guidelines for transfection ......................................... 26
Transfect cells cultured in supplemented Grace’s Medium ...................... 27
Transfect cells cultured in serum-free medium ................................ 28
Isolate P0 virus stock ........................................................... 28
Introduction ............................................................... 28
Characteristics of infected cells ............................................. 29
Prepare the P0 virus stock .................................................. 29
Store the virus stocks ...................................................... 29
Virus stock applications .................................................... 30
Amplify baculovirus stock ....................................................... 30
Introduction ............................................................... 30
Required materials ........................................................ 30
Multiplicity of infection (MOI) ................................................ 30
Contents
4
Bac-to-Bac
™
Baculovirus Expression System User Guide
Important considerations ................................................... 31
Amplify P0 virus stock in a 1-L flask .......................................... 31
Scale up the amplification procedure ......................................... 31
Generate high-titer stocks from frozen master stock ........................... 31
Perform a plaque assay ......................................................... 32
Introduction ............................................................... 32
Experimental outline ....................................................... 32
Factors affecting virus titer ................................................. 32
Required materials ........................................................ 32
Prepare the plaquing medium ............................................... 33
Perform plaque assay ...................................................... 34
Neutral Red staining ....................................................... 35
Prepare Neutral Red agarose overlay (for use on Day 4) ........................ 35
Prepare Neutral Red stain (for use on Day 7–10 prior to counting plaques) ........ 35
Calculate the titer ......................................................... 36
What you should see ....................................................... 36
Plaque purification ......................................................... 36
Express recombinant protein .................................................... 37
Introduction ............................................................... 37
Positive control ............................................................ 37
Guidelines for expression ................................................... 37
Calculate virus volumes .................................................... 38
Optimize expression ....................................................... 38
Harvest baculovirus infected insect cells ..................................... 38
Analyze recombinant protein .................................................... 39
Introduction ............................................................... 39
Protease inhibitors ......................................................... 39
Prepare cell lysates ........................................................ 40
Detect recombinant protein ................................................. 40
Assay for β-glucuronidase .................................................. 40
Assay for CAT ............................................................. 40
Remove the 6x His tag using AcTEV™ Protease ................................ 40
■APPENDIX A Troubleshooting ......................................... 41
Cloning into pFastBac™ vectors .................................................. 41
Recombinant bacmid DNA generation ............................................ 42
Bacmid DNA isolation .......................................................... 43
Insect cells transfection ........................................................ 44
Protein expression ............................................................. 44
Contents
Bac-to-Bac
™
Baculovirus Expression System User Guide
5
■APPENDIX B Vectors................................................... 46
pFastBac™ vectors ............................................................. 46
pFastBac™1................................................................... 46
Cloning considerations ..................................................... 46
Leading sequences ........................................................ 47
Features of the vector ...................................................... 47
Multiple cloning site of pFastBac™1.......................................... 48
pFastBac™ 1 vector map .................................................... 48
pFastBac™HT A, B, and C ........................................................ 49
Introduction ............................................................... 49
Cloning considerations ..................................................... 49
Leading sequences ........................................................ 49
Features of the vector ...................................................... 49
Multiple cloning site of pFastBac™HT A ....................................... 50
Multiple cloning site of pFastBac™HT B ....................................... 51
Multiple cloning site of pFastBac™HT C ....................................... 52
pFastBac™HT vector map ................................................... 53
pFastBac™ Dual ................................................................ 53
Introduction ............................................................... 53
Cloning considerations ..................................................... 54
Leading sequences ........................................................ 54
Features of the vector ...................................................... 54
Multiple cloning site downstream of the PH promoter .......................... 55
Multiple cloning site downstream of the p10 promoter .......................... 55
pFastBac™ Dual vector map ................................................. 56
pFastBac™ 1-Gus .............................................................. 56
Description ............................................................... 56
pFastBac™ 1-Gus Control Vector map ........................................ 57
pFastBac™ HT-CAT ............................................................. 57
Description ............................................................... 57
pFastBac™HT-CAT vector map ............................................... 58
pFastBac™ Dual-Gus/CAT ....................................................... 58
Description ............................................................... 58
pFastBac™ Dual-Gus/CAT vector map ........................................ 59
■APPENDIX C Supporting protocols .................................... 60
Antibiotic stock solutions ....................................................... 60
IPTG (200 mg/mL) stock solution ................................................. 60
Bluo-gal (20 mg/mL) stock solution .............................................. 60
LB (Luria-Bertani) Medium ...................................................... 61
LB (Luria-Bertani) Plates ....................................................... 61
Contents
6
Bac-to-Bac
™
Baculovirus Expression System User Guide
■APPENDIX D Bacmid DNA Isolation Using PureLink™ HiPure
Maxiprep Kit .............................................................. 62
Introduction ................................................................... 62
Grow bacmid DNA stock ........................................................ 62
Before Starting ................................................................ 63
Equilibrate the column .......................................................... 63
Prepare the cell lysate .......................................................... 63
Bind and wash the DNA ......................................................... 64
Elute and precipitate the DNA ................................................... 64
■APPENDIX E Ordering information .................................... 66
Additional products ............................................................ 66
Insect cell culture products ..................................................... 67
Purifying recombinant fusion proteins ............................................ 68
Nalgene flasks and tissue culture plates .......................................... 68
■APPENDIX F Safety ..................................................... 69
Chemical safety ................................................................ 70
Biological hazard safety ......................................................... 71
■APPENDIX G Documentation and support ............................ 72
Customer and technical support ................................................. 72
Limited product warranty ....................................................... 72
Contents
Bac-to-Bac
™
Baculovirus Expression System User Guide
7
Product information
IMPORTANT! Before using this product, read and understand the information in the
“Safety” appendix in this document.
Product description
The Bac-to-Bac™ Baculovirus Expression System enables rapid and ecient
generation of recombinant baculovirus. The system takes advantage of the site-
specic transposition properties of the Tn7 transposon to simplify and enhance the
process of generating recombinant bacmid DNA.
Kit contents and storage
Types of products
Product Amount Cat. No.
Bac-to-Bac™ Baculovirus Expression System 1 kit, 3 boxes 10359-016
Bac-to-Bac™ Vector Kit 1 kit, 1 box 10360014
Bac-to-Bac™ HT Vector Kit 1 kit, 1 box 10584027
pFastBac™ Dual Expression Vector 1 kit, 1 box 10712-024
The pFastBac™ Dual Expression Vector and the Bac-to-Bac™ Vector and HT Vector Kits
are each shipped in one box, whereas the Bac-to-Bac™ Baculovirus Expression System
is shipped in three boxes. On receipt, store each box as described. All reagents are
guaranteed for at least six months when stored properly.
Shipping and
storage
8
Bac-to-Bac
™
Baculovirus Expression System User Guide
Each product includes specic pFastBac™ vectors and a corresponding expression
control, according to the following table.
Product pFastBac™ vector Amount Storage
Bac-to-Bac™ Baculovirus
Expression System
pFastBac™1 20 µL at 0.5 µg/µL in TE, pH
8.0[1] (10 µg total)
2 to 8°C
pFastBac™-Gus 20 µL at 0.2 ng/µL in TE, pH 8.0
(4 ng total)
Bac-to-Bac™ Vector Kit pFastBac™1 20 µL at 0.5 µg/µL in TE, pH 8.0
(10 µg total)
pFastBac™-Gus 20 µL at 0.2 ng/µL in TE, pH 8.0
(4 ng total)
Bac-to-Bac™ HT Vector Kit pFastBac™HT A
pFastBac™HT B
pFastBac™HT C
20 µL each at 0.5 µg/µL in TE,
pH 8.0 (10 µg total of each
vector)
pFastBac™HT-CAT 15 µL at 1 ng/µL in TE, pH 8.0
(15 ng total)
pFastBac™ Dual pFastBac™ Dual 20 µL at 0.5 µg/µL in TE, pH 8.0
(10 µg total)
pFastBac™ Dual-Gus/CAT 20 µL at 0.2 ng/µL in TE, pH 8.0
(4 ng total)
[1] TE buffer, pH 8.0: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.
MAX Efficiency™ DH10Bac™ Competent
E. coli
Box 2
Reagent Composition Amount Storage
MAX Efficiency™ DH10Bac™ Competent
E. coli
—
4 kits
(5 × 100 µL
per kit) −80°C
pUC19 Control DNA 0.01 µg/mL in 5 mM Tris-HCl,
0.5 mM EDTA, pH 8.0 100 µL
MAX Efficiency™ DH10Bac™ Competent
E. coli
Genotype F−
mcr
A Δ(
mrr
-
hsd
RMS-
mcr
BC) ϕ80
lac
ZΔM15 ΔlacX74
rec
A1
end
A1
ara
D139
Δ(
ara
,
leu
)7697
gal
U
gal
K λ−
rps
L
nup
G/bMON14272/pMON7124
Transformation efficiency 1 × 108 cfu/μg of DNA
pFastBac™ vectors
Product information
Kit contents and storage
Bac-to-Bac
™
Baculovirus Expression System User Guide
9
ExpiFectamine™ Sf Transfection Reagent
Box 3
Reagent Amount Storage
ExpiFectamine™ Sf Transfection Reagent 1 mL 4°C (do not freeze)
The Bac-to-Bac™ Baculovirus Expression System
The Bac-to-Bac™ Baculovirus Expression System provides a rapid and highly eective
method to generate recombinant baculoviruses based on site-specic transposition of
an expression cassee into a baculovirus shule vector (bacmid) propagated in E. coli.
The major components of the Bac-to-Bac™ Baculovirus Expression System include:
• A choice of pFastBac™ donor plasmid that allows generation of an expression
construct containing the gene of interest under the control of a baculovirus-
specic promoter.
• An E. coli host strain, DH10Bac™ , that contains a bacmid and a helper plasmid,
and allows generation of a recombinant bacmid following transposition of the
pFastBac™ expression construct.
• ExpiFectamine™ Sf Transfection Reagent for fast and ecient transfection of
insect cells to generate recombinant baculovirus.
• A control expression plasmid containing the Gus and or CAT gene that allows
production of a recombinant baculovirus which, when used to infect insect cells,
expresses the Arabidopsis thaliana β-glucuronidase and or chloramphenicol acetyl-
transferase.
The rst major component of the System is a pFastBac™ vector into which your gene
of interest will be cloned.
Expression of the gene of interest is controlled by the Autographa californica multiple
nuclear polyhedrosis virus (AcMNPV) polyhedrin (PH) or p10 promoter for high-level
expression in insect cells. This expression cassee is anked by the left and right arms
of Tn7, and contains a gentamicin resistance gene and an SV40 polyadenylation signal
to form a mini Tn7.
The second major component of the System is the DH10Bac™ E. coli strain that is used
as the host for your pFastBac™ construct containing your gene of interest. DH10Bac™
cells contain a bacmid with a mini-aTn7 target site and a helper plasmid.
Once the pFastBac™ expression plasmid (the "donor plasmid") is transformed into
DH10Bac™ cells, transposition occurs between the mini-Tn7 element on the pFastBac™
vector and the mini-aTn7 target site on the bacmid to generate a recombinant
bacmid. This transposition reaction occurs in the presence of transposition proteins
supplied by the helper plasmid.
Once you have performed the transposition reaction, you will isolate the high
molecular weight recombinant bacmid DNA and transfect the bacmid DNA into
insect cells using the ExpiFectamine™ Sf Transfection Reagent to generate a
recombinant baculovirus that can be used for preliminary expression experiments.
System
components
pFastBac™ vector
DH10Bac™
E. coli
Product information
The Bac-to-Bac
™
Baculovirus Expression System
10
Bac-to-Bac
™
Baculovirus Expression System User Guide
After the baculovirus stock is amplied and titered, the high-titer stock can be used to
infect insect cells for large-scale expression of the recombinant protein of interest.
The baculovirus shule vector (bacmid), bMON14272 (136 kb), present in DH10Bac™
E. coli contains:
• A low-copy number mini-F replicon.
• Kanamycin resistance marker.
• A segment of DNA encoding the lacZ alpha peptide gene from a pUC-based
cloning vector into which the aachment site for the bacterial transposon, Tn7
(mini-aTn7) has been inserted. Insertion of the mini-aTn7 aachment site does
not disrupt the reading frame of the lacZ alpha peptide gene.
The bacmid propagates in DH10Bac™ E. coli as a large plasmid that confers resistance
to kanamycin and can complement a lacZ gene deletion present on the chromosome to
form colonies that are blue (lac+) in the presence of a chromogenic substrate such as
Bluo-gal or X-gal and the inducer, IPTG.
Recombinant bacmids are generated by transposing a mini-Tn7 element from a
pFastBac™ donor plasmid to the mini-aTn7 aachment site on the bacmid. The Tn7
transposition functions are provided by a helper plasmid.
DH10Bac™ E. coli also contain the helper plasmid, pMON7124 (13.2 kb), which
encodes the transposase and confers resistance to tetracycline. The helper plasmid
provides the Tn7 transposition function in trans.
ExpiFectamine™ Sf Transfection Reagent is a next-generation proprietary cationic lipid
transfection reagent for ecienct transfection of plasmid DNA and baculovirus
production in insect cells. ExpiFectamine™ Sf is a component of the ExpiSf™
Expression System (Cat. No. A38841, A39112, or A39111). To learn more about this
reagent and the ExpiSf™ Expression System for superior protein yields, see
thermosher.com/expisf.
Baculovirus
shuttle vector
Helper plasmid
ExpiFectamine™ Sf
Transfection
Reagent
Product information
The Bac-to-Bac
™
Baculovirus Expression System
Bac-to-Bac
™
Baculovirus Expression System User Guide
11
pFastBacTM TOPO
donor plasmid
Donor
Tn7R
Recombinant
Donor Plasmid
Tn7L
P
PH
Helper
TetR
AmpR
Donor
GmR
lacZ mini-attTn7
KanR
Bacmid
Gene of interest
Bacmid
Helper
Transposition
Antibiotic selection
Transformation
Mini-prep of
high molecular
weight DNA
Competent DH10BacTM E.coli cells
Determine virus
titer by plaque
assay or FACS
Infection of
insect cells
Recombinant
baculovirus
particles
Recombinant baculovirus
particles
Transfection of insect cells
with
Recombinant bacmid DNA
Recombinant gene expression
or
virus amplification
ExpifectamineTM Sf Reagent
E.coli (lacZ-) containing
recombinant bacmid
Figure 1 Generation of recombinant baculovirus and the expression of your gene of
interest using the Bac-to-Bac™ Baculovirus Expression System
Diagram of the
Bac-to-Bac™
System
Product information
The Bac-to-Bac
™
Baculovirus Expression System
12
Bac-to-Bac
™
Baculovirus Expression System User Guide
pFastBac™ donor plasmid
pFastBac™ recombinant
E.coli colonies with recombinant bacmid
Verified E.coli colonies with recombinant bacmid
Recombinant bacmid
P0 recombinant baculovirus stock >106 pfu/mL or 107 ivp/mL
P1 recombinant baculovirus stock >108 pfu/mL or 109 ivp/mL
Protein expression
Workflow
Product information
The Bac-to-Bac
™
Baculovirus Expression System
Bac-to-Bac
™
Baculovirus Expression System User Guide
13
Methods
Culture insect cells
We recommend using Spodoptera frugiperda Sf9 or Sf21 insect cells as the host for
recombinant baculovirus production. You can also use the high-density, suspension
ExpiSf9™ cell line, a component of the ExpiSf™ Expression System, to produce your
recombinant baculovirus. For additional information on baculovirus generation and
protein expression using the ExpiSf™ Expression System, go to thermosher.com/
expisf. Before you start your transfection and expression experiments, ensure to have
cultures of Sf9 or Sf21 cells growing and have frozen master stocks available. Sf9 or
Sf21 cells and cell culture reagents are available separately.
Note: High Five™ cells and Mimic™ Sf9 insect cells are appropriate for expression
only.
Insect cells may be cultured under serum-free conditions. We recommend using
Sf-900™ II SFM or Sf-900™ III SFM. If you are using the ExpiSf™ Expression System,
use ExpiSf™ CD Medium for cell culture.
Both Sf-900™ II SFM and Sf-900™ III SFM are protein-free media optimized for the
growth and maintenance of Sf9 and Sf21 cells, as well as for the large-scale production
of recombinant proteins expressed using the Bac-to-Bac™ System.
ExpiSf™ CD Medium is an innovative chemically-dened, yeastolate-free, animal
origin-free, serum-free, protein-free formulation that has been optimized for the
growth and maintenance of high-density suspension ExpiSf9™ cells, and for large-
scale production of recombinant proteins expressed using the Bac-to-Bac™ System. For
more information, see thermosher.com/expisf.
For guidelines and detailed information on insect cell culture, such as in the following
list, refer to the Guide to Baculovirus Expression Vector System (BEVS) and Insect Cell
Culture Techniques at thermosher.com.
• Maintaining and passaging insect cells in adherent and suspension culture
• Freezing cells
• Using serum-free medium (includes protocols to adapt cells to serum-free
medium)
• Scaling up cell culture
Introduction
Serum-free
medium
Insect cell culture
reference guide
14
Bac-to-Bac
™
Baculovirus Expression System User Guide
Insect cells are sensitive to environmental factors. In addition to chemical and
nutritional culture factors, physical factors can also aect insect cell growth. Therefore
optimization is required to maximize cell growth. Consider the following when
culturing insect cells:
•Temperature: The optimal range to grow and infect cultured insect cells is 27°C
to 28°C.
•pH: A range of 6.1 to 6.4 works well for most culture systems. Sf-900™ II SFM and
Sf-900™ III SFM maintain a pH in this range under conditions of normal air and
open-capped culture systems.
•Osmolality: The optimal osmolality of medium for use with lepidopteran cell
lines is 345 to 380 mOsm/kg.
•Aeration: Insect cells require passive oxygen diusion for optimal growth and
recombinant protein expression. Active or controlled oxygenated systems require
dissolved oxygen at 10% to 50% of air saturation.
•Shear forces: Suspension culture generates mechanical shear forces. Growing
insect cells in serum-containing media (10% to 20% FBS) generally provides
adequate protection from cellular shear forces. If you are growing insect cells in
serum-free conditions other than Sf-900™ II SFM or Sf-900™ III SFM,
supplementation with a shear force protectant such as Pluronic™ F-68 can be
required.
You need log-phase Sf9 or Sf21 cells with >95% viability to perform a successful
transfection. To determine how many cells you need for transfection, see
“Transfection conditions“ on page 26.
Generate recombinant pFastBac™ donor plasmid
To generate a recombinant plasmid containing your gene of interest for use in the Bac-
to-Bac™ Baculovirus Expression System, use restriction enzyme digestion and ligation
to clone your gene into one of the pFastBac™ vectors. For guidelines to help design
your cloning strategy, and for information on vector maps, multiple cloning sites, and
features, see Appendix B, “Vectors“.
Transform the cloned-insert ligation reaction into E. coli and select for ampicillin-
resistant transformants. Use any recA, endA E. coli strain, such as TOP10, DH10B™, or
DH5α™ for transformation. Do not transform the ligation reaction into DH10Bac™
cells.
Table 1 Chemically competent
E.Coli
cells
Item Quantity Cat. No.
One Shot™ TOP10 Chemically Competent
E. coli
20 × 50 µL C404003
One Shot™MAX Efficiency™ DH10B™ T1 Phage-Resistant
Cells 20 × 50 µL 12331013
One Shot™MAX Efficiency™ DH5α™-T1R Competent Cells 20 × 50 µL 12297016
General cell
culture guidelines
Cells for
transfection
Introduction
Select
E. coli
host
Methods
Generate recombinant pFastBac
™
donor plasmid
Bac-to-Bac
™
Baculovirus Expression System User Guide
15
You may use any method of choice to transform E. coli. Chemical transformation is the
most convenient method, while electroporation is the most ecient and method of
choice for large plasmids. To select for transformants, use LB agar plates containing
100 µg/mL ampicillin.
1. Pick 10 transformants and culture them overnight in LB or S.O.B. containing
100 µg/mL ampicillin.
2. Isolate the plasmid DNA using your method of choice.
We recommend using the PureLink™HiPure Plasmid DNA Miniprep Kit to
purify high quality plasmid DNA from your E. coli transformants.
3. Analyze the plasmids by restriction analysis to conrm the presence and correct
orientation of the insert. Use a restriction enzyme or a combination of enzymes
that cut once in the vector and once in the insert.
Requires the Platinum™ SuperFi™ PCR Master Mix or equivalent.
1. For each sample, aliquot 48 µL of Platinum™ SuperFi™ PCR Master Mix into a
0.5-mL microcentrifuge tube, then add 1 µL each of the forward and reverse PCR
primer.
2. Pick up to 10 colonies and resuspend them individually in 50 µL of the PCR
Master Mix containing primers.
Make a patch plate to preserve the colonies for further analysis.
3. Incubate the reaction for 10 minutes at 94°C to lyse the cells and inactivate
nucleases.
4. Amplify for 20 to 30 cycles.
5. For the nal extension, incubate at 72°C for 10 minutes, then store at 4°C.
6. Visualize by agarose gel electrophoresis.
Sequence the construct to conrm the gene of interest is in the correct orientation for
expression. If the gene was cloned into one of the pFastBac™HT vectors, verify that the
gene is cloned in frame with the N-terminal tag.
1. Streak the original colony out for single colony on LB plates containing
100 µg/mL ampicillin.
2. Isolate a single colony and inoculate into 1–2 mL of LB containing 100 µg/mL
ampicillin.
3. Grow until the culture reaches the stationary phase.
4. Mix 0.85 mL of culture with 0.15 mL of sterile glycerol, then transfer to a
cryovial.
5. Store at –80°C.
Transformation
method
Analyze the
transformants
Analyze the
transformants by
PCR
Sequencing
Make a glycerol
stock for long-
term storage
Methods
Generate recombinant pFastBac
™
donor plasmid
16
Bac-to-Bac
™
Baculovirus Expression System User Guide
Generate recombinant bacmid DNA: transform DH10Bac™
E. coli
The pFastBac™ construct containing your gene of interest in the correct orientation is
used to transform puried plasmid DNA into MAX Eciency™ DH10Bac™
Competent Cells for transposition into the bacmid. Then, blue/white selection
identies colonies containing the recombinant bacmid.
Guidelines and instructions to transform DH10Bac™ E. coli using the pFastBac™
construct are provided in this section.
Each pFastBac™ plasmid is supplied with a corresponding control plasmid for use as a
positive transfection and expression control (see the following table). We recommend
including the corresponding control plasmid in your DH10Bac™ transformation
experiment. For maps and a description of the features of each control plasmid, see
Appendix B: Vectors.
pFastBac™ Vector Control Plasmid
pFastBac™1 pFastBac™ 1-Gus
pFastBac™HT pFastBac™HT-CAT
pFastBac™ Dual pFastBac™ Dual-Gus/CAT
• Your puried pFastBac™ construct (200 pg/mL in TE, pH 8.0)
• Positive expression control plasmid (as a transposition control)
• MAX Eciency™ DH10Bac™ Competent Cells (use 1 tube per transformation)
• pUC19 (as a transformation control)
• LB agar plates containing kanamycin, gentamicin, tetracycline, Bluo-gal, and
IPTG (3 freshly prepared plates per transformation, see “Recommendation“ on
page 18 and Appendix C, “Supporting protocols“)
• LB agar plate containing 100 mg/mL ampicillin (for pUC19 transformation
control)
• SOC Medium
• 15-mL round-boom polypropylene tubes
• 42°C water bath
• 37°C shaking and non-shaking incubator
Introduction
Positive control
Required
materials
Methods
Generate recombinant bacmid DNA: transform DH10Bac
™
E. coli
Bac-to-Bac
™
Baculovirus Expression System User Guide
17
Prepare LB agar plates containing:
• 50 µg/mL kanamycin
• 7 µg/mL gentamicin
•10 µg/mL tetracycline
• 100 µg/mL Bluo-gal
• 40 µg/mL IPTG
Note: Use Bluo-gal instead of X-gal for blue/white selection as Bluo-gal generally
produces a darker blue color than X-gal.
To order antibiotics, Bluo-gal, and IPTG, see Appendix E, “Ordering information“,
and for instructions to prepare plates, see “LB (Luria-Bertani) Plates“ on page 61. We
recommend using Luria Broth Base (see “LB (Luria-Bertani) Medium“ on page 61)
instead of Lennox L (LB) as the color intensity and number of colonies that are
obtained on Lennox L plates is reduced.
Each transformation requires one vial of DH10Bac™ competent cells and three
selective plates.
• Equilibrate a water bath to 42°C.
• Warm selective plates at 37°C for 30 minutes.
• Warm the SOC Medium to room temperature.
• Pre-chill one 15-mL round-boom polypropylene tube for each transformation.
1. Thaw on ice one vial of MAX Eciency™ DH10Bac™ Competent Cells for each
transformation.
2. For each transformation, gently mix, then transfer 100 µL of the DH10Bac™ cells
into a pre-chilled, 15-mL round-boom polypropylene tube.
3. Add the plasmid DNA to the cells, according to the following table, then mix
gently.
Note: Do not pipet up and down to mix.
Table 2
Plasmid DNA Amount to add
Your recombinant pFastBac™ construct 1 ng (5 µL)
pFastBac™ control plasmid 1 ng
pUC19 control 50 pg (5 µL)
4. Incubate the cells on ice for 30 minutes.
5. Heat-shock the cells for 45 seconds at 42°C without shaking.
6. Immediately transfer the tubes to ice and chill for 2 minutes.
7. Add 900 µL of room temperature SOC Medium.
Recommendation
Prepare for
transformation
Transform
DH10Bac™
E. coli
Methods
Generate recombinant bacmid DNA: transform DH10Bac
™
E. coli
18
Bac-to-Bac
™
Baculovirus Expression System User Guide
8. Transform cells according to the following table.
To transform Description
pFastBac™Shake tubes at 37°C at 225 rpm for 4 hours
pUC19 Shake tube at 37°C at 225 rpm for 1 hour
9. Prepare dilutions according to the following table.
For plasmid Description
pFastBac™Prepare 10-fold serial (10−1 to 10−3) dilutions of the cells with SOC
Medium, then plate 100 µL of each dilution on an LB agar plate
containing:
• 50 µg/mL kanamycin
• 7 µg/mL gentamicin
• 10 µg/mL tetracycline
• 100 µg/mL Bluo-gal
• 40 µg/mL IPTG
pUC19 Dilute the cells 1:100 with SOC Medium, then plate 100 µL of the
dilution on an LB agar plate containing 100 µg/mL ampicillin.
10. Incubate plates for 24–48 hours at 37°C.
IMPORTANT! Insertion of the mini-Tn7 into the mini-aTn7 aachment site on
the bacmid disrupts the expression of the LacZ peptide. Therefore, colonies
containing the recombinant bacmid are white. Blue colonies contain unaltered
bacmid.
11. Pick white colonies for analysis.
Note: True white colonies tend to be large, therefore avoid false positives by
selecting the largest, most isolated white colonies. Avoid picking colonies that
appear gray or that are darker in the center as they can contain a mixture of cells
with empty bacmid and recombinant bacmid.
1. Pick up to 10 white colonies, then restreak them on fresh LB agar plates
containing:
• 50 µg/mL kanamycin
• 7 µg/mL gentamicin
• 10 µg/mL tetracycline
• 100 µg/mL Bluo-gal
• 40 µg/mL IPTG
2. Incubate the plates overnight at 37°C.
3. Pick a white colony, then inoculate a liquid culture that contains:
• 50 µg/mL kanamycin
• 7 µg/mL gentamicin
• 10 µg/mL tetracycline
Verify the
phenotype
Methods
Generate recombinant bacmid DNA: transform DH10Bac
™
E. coli
Bac-to-Bac
™
Baculovirus Expression System User Guide
19
4. Isolate recombinant bacmid DNA, see “Isolate recombinant bacmid DNA“ on
page 20.
Note: For increased recombinant bacmid yield, use the procedure for the
PureLink™ HiPure Plasmid Maxiprep Kit, see Appendix D, “Bacmid DNA
Isolation Using PureLink™ HiPure Maxiprep Kit“.
Note: Bacmid DNA must be clean and free from phenol and sodium chloride as
contaminants can kill insect cells, and salt interferes with lipid complexing,
which decreases the transfection eciency.
5. Analyze the recombinant bacmid DNA by PCR to verify successful transposition
to the bacmid, see “Analyze recombinant bacmid DNA by PCR“ on page 22.
Isolate recombinant bacmid DNA
The PureLink™HiPure Plasmid DNA Miniprep Kit allows you to purify high-quality
bacmid DNA from DH10Bac™ E. coli (see Appendix E, “Ordering information“). The
isolated bacmid DNA is appropriate for use in insect cell transfections.
Note: We do not recommend the PureLink™ HiPure Precipitator Module or the
PureLink™ HiPure Plasmid Filter Mini/Midi/Maxiprep Kits for isolating bacmid DNA.
Note: When using the ExpiSf™ Expression System, we recommend isolating bacmid
DNA using the PureLink™ HiPure Plasmid Maxiprep Kit (Cat. No. K2100-06). This
will ensure there is enough, high-quality bacmid DNA for baculovirus generation
using suspension-based transfection. Follow instructions in PureLink™ HiPure Plasmid
Purication Kits User Guide, (Pub. No. MAN0000486) using the MaxiPrep low-copy
plasmid instructions.
• Prepare LB medium containing:
– 50 µg/mL kanamycin
–7 µg/mL gentamicin
– 10 µg/mL tetracycline
Inoculate a single white bacterial colony into 2 mL of this LB medium, then
incubate the culture at 37°C in a shaking water bath at 250 rpm overnight.
• Verify that RNase A is added to the Resuspension Buer (R3).
• Ensure the Lysis Buer (L7) contains no precipitates.
1. Place the PureLink™ HiPure Mini column on the PureLink™ Nucleic Acid
Purication Rack.
2. Add 2 mL of Equilibration Buer (EQ1) to the column.
3. Allow the solution in the column to drain by gravity ow.
Introduction
Before you begin
Equilibrate the
column
Methods
Isolate recombinant bacmid DNA
20
Bac-to-Bac
™
Baculovirus Expression System User Guide
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