Thermo Fisher Scientific Bac-to-Bac Baculovirus Expression System User guide

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.

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 ecient

generation of recombinant baculovirus. The system takes advantage of the site-

specic 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 specic 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 eective

method to generate recombinant baculoviruses based on site-specic transposition of

an expression cassee into a baculovirus shule 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-

specic 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 ecient 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 cassee 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-aTn7 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-aTn7 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 amplied 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 shule 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 aachment site for the bacterial transposon, Tn7

(mini-aTn7) has been inserted. Insertion of the mini-aTn7 aachment 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-aTn7 aachment 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 ecienct 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

thermosher.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 thermosher.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-dened, 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 thermosher.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 thermosher.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 aect 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 diusion 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 ecient 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 conrm 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 conrm 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 puried plasmid DNA into MAX Eciency™ DH10Bac™

Competent Cells for transposition into the bacmid. Then, blue/white selection

identies 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 puried pFastBac™ construct (200 pg/mL in TE, pH 8.0)

• Positive expression control plasmid (as a transposition control)

• MAX Eciency™ 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-boom 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-boom polypropylene tube for each transformation.

1. Thaw on ice one vial of MAX Eciency™ 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-boom 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-aTn7 aachment 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 eciency.

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

Purication 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 Buer (R3).

• Ensure the Lysis Buer (L7) contains no precipitates.

1. Place the PureLink™ HiPure Mini column on the PureLink™ Nucleic Acid

Purication Rack.

2. Add 2 mL of Equilibration Buer (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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Thermo Fisher Scientific Bac-to-Bac Baculovirus Expression System User guide

Thermo Fisher Scientific Bac-to-Bac Baculovirus Expression System User guide

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