September 2000
PolyFect
®
Transfection Reagent
Handbook
For optimized transfection of COS-7, NIH/3T3, HeLa,
293, and CHO cells
1015222HBPolyFect_0900WW.qxd 28.08.2000 17:03 Uhr Seite 1
Trademarks
Patented or patent-pending technology and/or registered or registration-pending trademark of QIAGEN: QIAGEN
®
,
EndoFree
®
, PolyFect
®
, HiSpeed
™
, QIAfilter
™
, SuperFect
™
, Effectene
™
.
© 2000 QIAGEN, all rights reserved.
QIAGEN Distributors
Please see the last page for contact information for your local QIAGEN distributor.
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ABN 75 072 382 944 Orders 03-9489-3666 • Fax 03-9489-3888 • Technical 03-9489-3666
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www.qiagen.com
QIAGEN Companies
QIAGEN Worldwide
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PolyFect Transfection Reagent Handbook 09/2000 3
Contents
Kit Contents 5
Storage and Stability 5
Quality Control 5
Technical Assistance 5
Introduction 6
The PolyFect Principle 6
General Guidelines 7
Cell culture 7
Effect of serum 7
Vector construct 7
Plasmid DNA quality 7
Ratio of PolyFect Reagent to DNA 8
Transfection of oligonucleotides or large vector constructs 8
Protocol for Transient Transfection of COS-7, NIH/3T3, and CHO Cells 10
Protocol for Transient Transfection of HeLa Cells 12
Protocol for Transient Transfection of 293 Cells 14
Troubleshooting Guide 16
Appendix A: Composition of Buffers 19
Appendix B: Background Information 19
Transfection Principle 19
Transient transfection 19
Stable transfection 19
Primary cells and cell lines 20
Adherent cells 20
Suspension cells 20
Primary cell culture 20
Finite cell line 20
Continuous cell line 20
Transfection considerations 20
Media and supplements 20
Serum 21
Transfection methods 21
Plasmid DNA quality 21
1015222HBPolyFect_0900WW.qxd 28.08.2000 17:03 Uhr Seite 3
Genetic Reporter Systems 21
Chloramphenicol acetyltransferase 22
Firefly luciferase 22
β-Galactosidase 22
Human growth hormone (hGH) 23
Green fluorescent protein 23
References 23
Product Use Limitations 24
Product Warranty and Satisfaction Guarantee 24
Ordering Information 25
QIAGEN International Distributors 27
PolyFect Transfection Reagent Handbook 09/20004
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PolyFect Transfection Reagent Handbook 09/2000 5
Kit Contents
1.0 ml of PolyFect Transfection Reagent (2 mg/ml), sufficient for 25–65 transfections in
60 mm dishes, or 50–100 transfections in 6-well plates.
Storage and Stability
PolyFect
®
Transfection Reagent is supplied as a ready-to-use solution and is shipped at ambient
temperature without loss in stability. However, it should be stored at 2–8°C upon arrival.
PolyFect Transfection Reagent is stable for 1 year at 2–8°C.
Quality Control
Endotoxin levels are ≤10 EU/ml determined using a Kinetic-QCL test (BioWhittaker, Inc.)
PolyFect Transfection Reagent is tested by transfection of pCMVβ (CLONTECH) into HeLa-S3
and COS-7 cells to ensure lot-to-lot consistency. Sterility tests guarantee absence of any
contaminating bacteria or fungi.
Technical Assistance
At QIAGEN we pride ourselves on the quality and availability of our technical support.
Our Technical Services Departments are staffed by experienced scientists with extensive
practical and theoretical expertise in molecular biology and the use of QIAGEN products.
If you have any questions or experience any difficulties regarding PolyFect Transfection
Reagent, or QIAGEN products in general, please do not hesitate to contact us.
QIAGEN customers are a major source of information regarding advanced or specialized
uses of our products. This information is helpful to other scientists as well as to the
researchers at QIAGEN. We therefore encourage you to contact us if you have any
suggestions about product performance or new applications and techniques.
For technical assistance and more information, please call one of the QIAGEN Technical
Service Departments or local distributors listed on the last page.
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PolyFect Transfection Reagent Handbook 09/20006
Introduction
PolyFect Transfection Reagent has been specifically developed and designed for
transfection of commonly used cell lines. PolyFect Reagent protocols offer optimal condi-
tions for transfection of COS-7, NIH/3T3, HeLa, 293, and CHO cells in three different
plate formats. All protocols in this handbook have been optimized for each particular cell
line, eliminating the need for optimization trials. In addition, the fast and easy protocols
eliminate the need for removal of the complexes or a medium change after transfection.
PolyFect Reagent combines excellent performance with a convenient transfection method.
PolyFect Transfection Reagent offers:
• Fast and easy protocol
• No removal of complex required
• Optimized protocols for COS-7, NIH/3T3, HeLa, 293, and CHO cells
• Transfection in the presence of serum
• Economical price
• High efficiency
The PolyFect Principle
PolyFect Transfection Reagent is an activated-dendrimer transfection reagent specifically
designed for the transfection of commonly used cell lines. PolyFect Reagent offers significant
advantages over many other transfection methods.
PolyFect Reagent possesses a defined spherical architecture, with branches radiating from
a central core and terminating at charged amino groups. PolyFect Reagent assembles
DNA into compact structures, optimizing the entry of DNA into the cell. PolyFect–DNA
complexes possess a net positive charge, which allows them to bind to negatively charged
receptors (e.g., sialylated glycoproteins) on the surface of eukaryotic cells. Once inside
the cell, PolyFect Reagent buffers the lysosome after it has fused with the endosome, leading
to pH inhibition of lysosomal nucleases. This ensures stability of PolyFect–DNA complexes
and the transport of intact DNA into the nucleus.
NOTE: The amounts of DNA and PolyFect Reagent given in the protocols represent optimal
conditions for transfection of the particular cell line. Therefore, we strongly recommend
using the optimized amounts of DNA and PolyFect Reagent given in the appropriate protocol.
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PolyFect Transfection Reagent Handbook 09/2000 7
General Guidelines
Transfection efficiencies depend on a variety of parameters. The factors below should be
taken into account when performing transfections.
Cell culture
A healthy cell culture lays the foundation for successful transfection. Different cells or cell
lines have very specific medium, serum, and supplement requirements. Low passage number
(<50 splitting cycles) ensures that the cell genotype has not become altered. Highest
transfection efficiencies are obtained using the confluence levels indicated in the appro-
priate protocol sections. We recommend subculturing cells 24 h before transfection. This
ensures normal cell metabolism and increases the likelihood of DNA uptake. Contamination
with bacteria (e.g., mycoplasma) and fungi should be avoided, since this can drastically
alter transfection results. Antibiotics can be included in the medium used for transfection
with PolyFect Reagent and during subsequent incubation for gene expression.
Effect of serum
In contrast to many liposome transfection reagents, the presence of serum during trans-
fection with PolyFect Reagent significantly increases transfection efficiencies. Therefore, we
recommend using the percentage of serum that cells have been adapted to in culture
during incubation of cells with transfection complexes. However, we do not recommend
using serum during complex formation between PolyFect Reagent and plasmid DNA, as
serum may inhibit complex formation.
Vector construct
The type of transfection vector (plasmid DNA, RNA, PCR products, oligonucleotides)
influences the transfection results. The configuration and size of the constructs determine
the efficiency of transfection. Transient transfection is most efficient with supercoiled plasmid
DNA. In stable transfection, linear DNA yields optimal integration of the DNA into the
host genome, but results in lower DNA uptake by the cells, relative to supercoiled DNA.
Plasmid DNA quality
Plasmid DNA quality strongly influences several transfection parameters such as efficiency,
reproducibility and toxicity, as well as interpretation of results. Therefore only plasmid
DNA of the highest purity should be used. DNA purified with QIAGEN, QIAfilter
™
and
HiSpeed
™
Plasmid Kits is well suited for transfection of most cell lines. For best results with
all cell lines, we recommend DNA purified with the EndoFree
®
Plasmid Kit. This kit quickly
and efficiently removes bacterial endotoxins during the plasmid purification procedure,
ensuring optimal transfection results.
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PolyFect Transfection Reagent Handbook 09/20008
Ratio of PolyFect Reagent to DNA
The overall charge of PolyFect–DNA complexes is determined by the ratio of PolyFect
Reagent to DNA. Optimal binding of PolyFect–DNA complexes to the negatively charged
groups (e.g., sialylated glycoproteins) on the cell surface requires a slightly positive net
charge. To achieve an optimal ratio of PolyFect Reagent (µl) to DNA (µg), we strongly
recommend using the optimized amounts of DNA and volumes of PolyFect Reagent
indicated in the appropriate protocol.
Transfection of oligonucleotides or large vector constructs
For the transfection of oligonucleotides or large vector constructs (>20 kb), we recommend
carrying out optimization trials to determine optimal amounts of DNA and PolyFect
Reagent to be used. The amounts of DNA and PolyFect Reagent given in the protocols can
be used as a starting point.
To determine the optimal amount of PolyFect Reagent, set up trial reactions using double
and half the protocol-recommended amount, keeping the DNA amount as given in the
protocol. In a second set of reactions, use double and half the protocol-recommended
amount of DNA, keeping the PolyFect Reagent amount as given in the protocol. By
following this optimization strategy you should be able to determine the optimal PolyFect
Reagent/DNA ratio for your transfection.
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PolyFect Transfection Reagent Handbook 09/2000 9
DNA solution
Add PolyFect
Transfection Reagent
Incubate 5–10 min at RT
Add medium
Pipet up and down 2x
Apply diluted
complexes to cells
V
ortex for 10 seconds
Incubate
Evaluate transfection efficiency
or
Select for stably transfected cells
Transfection Procedure for
PolyFect Transfection Reagent
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PolyFect Transfection Reagent Handbook 09/200010
Protocol
Protocol for Transient Transfection of COS-7, NIH/3T3,
and CHO Cells
The following protocol is optimized for transient transfection of COS-7, NIH/3T3, and CHO
cells in 60 mm dishes. Parameters for transfection using other culture formats are given in
Table 1.
IMPORTANT: To ensure optimal results we strongly recommend using the optimized
amounts of DNA and PolyFect Reagent given in the protocol and table below.
1. The day before transfection, seed 8 x 10
5
cells per 60 mm dish in 5 ml of appropriate
growth medium.
2. Incubate the cells at 37°C and 5% CO
2
in an incubator. The dishes should be 40–80%
confluent on the day of transfection.
3. Dilute 2.5 µg of DNA dissolved in TE buffer pH 7 to pH 8 (minimum DNA concentration:
0.1 µg/µl) with cell growth medium containing no serum, proteins, or antibiotics to
a total volume of 150 µl. Mix and spin down the solution for a few seconds to remove
drops from the top of the tube.
IMPORTANT: Serum, proteins, and antibiotics present during this step interfere with
complex formation and will significantly decrease transfection efficiency.
Note: Plasmid DNA quality strongly influences several transfection parameters such
as efficiency, reproducibility and toxicity, as well as interpretation of results. Therefore
only plasmid DNA of the highest purity should be used. DNA purified with QIAGEN,
QIAfilter and HiSpeed Plasmid Kits is well suited for transfection of most cell lines.
For best results with all cell lines, we recommend DNA purified with the EndoFree
Plasmid Kit. This kit quickly and efficiently removes bacterial endotoxins during the
plasmid purification procedure, ensuring optimal transfection results.
4. Add 15 µl of PolyFect Transfection Reagent to the DNA solution. Mix by pipetting up
and down 5 times, or by vortexing for 10 sec.
Note: It is not necessary to keep PolyFect Reagent on ice at all times. 10–15 min at
room temperature will not alter its stability.
5. Incubate samples for 5–10 min at room temperature (20–25°C) to allow complex
formation.
6. While complex formation takes place, gently aspirate the growth medium from the
dish, wash cells once with 4 ml PBS, and add 3 ml of fresh cell growth medium
(containing serum and antibiotics).
7. Add 1 ml of cell growth medium (containing serum and antibiotics) to the reaction
tube containing the transfection complexes. Mix by pipetting up and down twice, and
immediately transfer the total volume to the cells in the 60 mm dishes. Gently swirl
the dish to ensure uniform distribution of the complexes.
At this stage serum and antibiotics no longer interfere with transfection and will
significantly enhance the transfection efficiency of PolyFect Reagent.
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PolyFect Transfection Reagent Handbook 09/2000 11
Protocol
8. Incubate cells with the complexes at 37°C and 5% CO
2
to allow for gene expression.
Harvest cells and assay for reporter gene expression after an appropriate
incubation time.
For example, cells transfected with
β
-gal or cat reporter constructs are typically
incubated for 24–48 h after transfection to obtain maximal expression levels of the
reporter gene.
Table 1. Parameters for transient transfection of COS-7, NIH/3T3, and CHO cells in
different formats
Final Volume
volume Volume of Volume of
Volume of of medium medium to
No. of of diluted PolyFect to add add to
Culture cells to medium
‡
DNA DNA Reagent to cells
‡
complexes
‡
format seed (ml) (µg) (µl) (µl) (ml) (ml)
protocol step 1 1 3 3 4 6 7
6-well plate* 4 x 10
5
3.0 1.5 100 10 1.5 0.6
60 mm dish 8 x 10
5
5.0 2.5 150 15 3.0 1.0
100 mm dish
†
1.6 x 10
6
8.0 4.0 300 25 7.0 1.0
* When working with 35 mm dishes use the amounts stated for 6-well plates.
†
When working with 85 or 90 mm dishes use the amounts stated for 100 mm dishes.
‡
Medium containing serum and antibiotics.
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PolyFect Transfection Reagent Handbook 09/200012
Protocol
Protocol for Transient Transfection of HeLa Cells
The following protocol is optimized for transient transfection of HeLa cells in 60 mm dishes.
Parameters for transfection using other culture formats are given in Table 2. Please note
that for HeLa-S3 cells the optimal amounts of DNA and PolyFect Reagent differ from the
amounts given in the protocol below. If you work with HeLa-S3 cells, please call one of
the QIAGEN technical service departments (see inside front cover) or your local distributor
(see inside back cover) for an optimized protocol.
IMPORTANT: To ensure optimal results we strongly recommend using the optimized
amounts of DNA and PolyFect Reagent given in the protocol and table below.
1. The day before transfection, seed 8 x 10
5
cells per 60 mm dish in 5 ml of appropriate
growth medium.
2. Incubate the cells at 37°C and 5% CO
2
in an incubator. The dishes should be 40–80%
confluent on the day of transfection.
3. Dilute 3 µg of DNA dissolved in TE buffer pH 7 to pH 8 (minimum DNA concentration:
0.1 µg/µl) with cell growth medium containing no serum, proteins, or antibiotics to
a total volume of 150 µl. Mix and spin down the solution for a few seconds to remove
drops from the top of the tube.
IMPORTANT: Serum, proteins, and antibiotics present during this step interfere with
complex formation and will significantly decrease transfection efficiency.
Note: Plasmid DNA quality strongly influences several transfection parameters such
as efficiency, reproducibility and toxicity, as well as interpretation of results. Therefore
only plasmid DNA of the highest purity should be used. DNA purified with QIAGEN,
QIAfilter and HiSpeed Plasmid Kits is well suited for transfection of most cell lines.
For best results with all cell lines, we recommend DNA purified with the EndoFree
Plasmid Kit. This kit quickly and efficiently removes bacterial endotoxins during the
plasmid purification procedure, ensuring optimal transfection results.
4. Add 25 µl of PolyFect Transfection Reagent to the DNA solution. Mix by pipetting up
and down 5 times, or by vortexing for 10 sec.
Note: It is not necessary to keep PolyFect Reagent on ice at all times. 10–15 min at
room temperature will not alter its stability.
5. Incubate samples for 5–10 min at room temperature (20–25°C) to allow complex
formation.
6. While complex formation takes place, gently aspirate the growth medium from the
dish, wash cells once with 4 ml PBS, and add 3 ml of fresh cell growth medium
(containing serum and antibiotics).
1015222HBPolyFect_0900WW.qxd 28.08.2000 17:03 Uhr Seite 12
13
Protocol
PolyFect Transfection Reagent Handbook 09/2000
7. Add 1 ml of cell growth medium (containing serum and antibiotics) to the reaction
tube containing the transfection complexes. Mix by pipetting up and down twice, and
immediately transfer the total volume to the cells in the 60 mm dishes. Gently swirl
the dish to ensure uniform distribution of the complexes.
At this stage serum and antibiotics no longer interfere with transfection and will sig-
nificantly enhance the transfection efficiency of PolyFect Reagent.
8. Incubate cells with the complexes at 37°C and 5% CO
2
to allow for gene expression.
Harvest cells and assay for reporter gene expression after an appropriate incubation
time.
For example, cells transfected with
β
-gal or cat reporter constructs are typically
incubated for 24–48 h after transfection to obtain maximal expression levels of the
reporter gene.
Table 2. Parameters for transient transfection of HeLa cells in different formats
Final Volume
volume Volume of Volume of
Volume of of medium medium to
No. of of diluted PolyFect to add add to
Culture to cells medium
‡
DNA DNA Reagent to cells
‡
complexes
‡
format seed (ml) (µg) (µl) (µl) (ml) (ml)
protocol step 1 1 3 3 4 6 7
6-well plate* 4 x 10
5
3.0 1.5 100 12 1.5 0.6
60 mm dish 8 x 10
5
5.0 3.0 150 25 3.0 1.0
100 mm dish
†
1.6 x 10
6
8.0 6.0 300 50 7.0 1.0
* When working with 35 mm dishes use the amounts given for 6-well plates.
†
When working with 85 or 90 mm dishes use the amounts given for 100 mm dishes.
‡
Medium containing serum and antibiotics.
1015222HBPolyFect_0900WW.qxd 28.08.2000 17:03 Uhr Seite 13
Protocol
PolyFect Transfection Reagent Handbook 09/200014
Protocol for Transient Transfection of 293 Cells
The following protocol is optimized for transient transfection of 293 cells in 60 mm dishes.
Parameters for transfection using other culture formats are given in Table 3.
IMPORTANT: To ensure optimal results we strongly recommend using the optimized
amounts of DNA and PolyFect Reagent given in the protocol and table below.
1. The day before transfection, seed 1.2 x 10
6
cells per 60 mm dish in 5 ml of
appropriate growth medium.
2. Incubate the cells at 37°C and 5% CO
2
in an incubator. The dishes should be 40–80%
confluent on the day of transfection.
3. Dilute 4 µg of DNA dissolved in TE buffer pH 7 to pH 8 (minimum DNA concentration:
0.1 µg/µl) with cell growth medium containing no serum, proteins, or antibiotics to
a total volume of 150 µl. Mix and spin down the solution for a few seconds to remove
drops from the top of the tube.
IMPORTANT: Serum, proteins, and antibiotics present during this step interfere with
complex formation and will significantly decrease transfection efficiency.
Note: Plasmid DNA quality strongly influences several transfection parameters such
as efficiency, reproducibility and toxicity, as well as interpretation of results. Therefore
only plasmid DNA of the highest purity should be used. DNA purified with QIAGEN,
QIAfilter and HiSpeed Plasmid Kits is well suited for transfection of most cell lines.
For best results with all cell lines, we recommend DNA purified with the EndoFree
Plasmid Kit. This kit quickly and efficiently removes bacterial endotoxins during the
plasmid purification procedure, ensuring optimal transfection results.
4. Add 40 µl of PolyFect Transfection Reagent to the DNA solution. Mix by pipetting up
and down 5 times, or by vortexing for 10 sec.
Note: It is not necessary to keep PolyFect Reagent on ice at all times. 10–15 min at
room temperature will not alter its stability.
5. Incubate samples for 5–10 min at room temperature (20–25°C) to allow complex
formation.
6. While complex formation takes place, gently aspirate the growth medium from the
dish and add 3 ml of fresh cell growth medium (containing serum and antibiotics).
7. Add 1 ml of cell growth medium (containing serum and antibiotics) to the reaction
tube containing the transfection complexes. Mix by pipetting up and down twice, and
immediately transfer the total volume to the cells in the 60 mm dishes. Gently swirl
the dish to ensure uniform distribution of the complexes.
At this stage serum and antibiotics no longer interfere with transfection and will
significantly enhance the transfection efficiency of PolyFect Reagent.
1015222HBPolyFect_0900WW.qxd 28.08.2000 17:03 Uhr Seite 14
Protocol
PolyFect Transfection Reagent Handbook 09/2000 15
8. Incubate cells with the complexes at 37°C and 5% CO
2
to allow for gene expression.
Harvest cells and assay for reporter gene expression after an appropriate
incubation time.
For example, cells transfected with
β
-gal or cat reporter constructs are typically
incubated for 24–48 h after transfection to obtain maximal expression levels of the
reporter gene.
Table 3. Parameters for transient transfection of 293 cells in different formats
Final Volume
volume Volume of Volume of
of of medium medium to
No. of Volume of diluted PolyFect to add add to
Culture cells to medium
‡
DNA DNA Reagent to cells
‡
complexes
‡
format seed (ml) (µg) (µl) (µl) (ml) (ml)
protocol step 1 1 3 3 4 6 7
6-well plate* 6 x 10
5
3.0 2.0 100 20 1.5 0.6
60 mm dish 1.2 x 10
6
5.0 4.0 150 40 3.0 1.0
100 mm dish
†
2.4 x 10
6
8.0 8.0 300 80 7.0 1.0
* When working with 35 mm dishes use the amounts given for 6-well plates.
†
When working with 85 or 90 mm dishes use the amounts given for 100 mm dishes.
‡
Medium containing serum and antibiotics.
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16 PolyFect Transfection Reagent Handbook 09/2000
Troubleshooting Guide
This troubleshooting guide may be helpful in solving any problems that may arise. The
scientists in QIAGEN Technical Services are always happy to answer any questions you
may have about either the information and protocol(s) in this handbook or molecular biology
applications (see last page for contact information).
Observation Possible Cause Comments and suggestions
Low transfection PolyFect Reagent or The amounts of DNA and PolyFect Reagent
efficiency DNA amount is given in the protocols represent optimal
suboptimal conditions for the transfection of the particular
cell line. To ensure optimal results use the
amounts of PolyFect Reagent and DNA given in
the appropriate protocol. However, if you are
working with oligonucleotides or large vector
constructs (> 20 kb) please follow the guidelines
given on page 8 under “Transfection of
Oligonucleotides or large vector constructs”.
Incubation time for Different cell types achieve maximal expression
gene expression is levels at different times post-transfection. This
suboptimal should be kept in mind when determining the
length of incubation with the complexes. If the
time point of maximal expression is not known
for a particular cell line, a time course experi-
ment may be necessary.
Cell density at the If the cell density is too high during complex
time of PolyFect-DNA addition cells may not be at the optimal phase
complex addition is of growth. This can lead to insufficient uptake
too high of the complexes into the cells or to insufficient
expression of the gene of interest. Be sure to use
the confluence levels indicated in the appropriate
protocol.
Reporter assay Include positive controls to ensure that the
problem reporter assay is working properly.
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17PolyFect Transfection Reagent Handbook 09/2000
Observation Possible Cause Comments and suggestions
Poor DNA quality Plasmid DNA quality strongly influences several
transfection parameters such as efficiency,
reproducibility and toxicity, as well as interpre-
tation of results. Therefore only plasmid DNA of
the highest purity should be used. DNA purified
with QIAGEN, QIAfilter and HiSpeed Plasmid
Kits is well suited for transfection of most cell
lines. For best results with all cell lines, we
recommend DNA purified with the EndoFree
Plasmid Kit. This kit quickly and efficiently
removes bacterial endotoxins during the
plasmid purification procedure, ensuring optimal
transfection results.
Excessive cell Amount of PolyFect The amounts of DNA and PolyFect Reagent given
death Reagent or DNA is in the protocols represent optimized conditions
too high for the transfection of the respective cell line. To
ensure optimal results use the amounts of Poly-
Fect Reagent and DNA given in the appropriate
protocol.
Cells are stressed In general, avoid stressing cells with temperature
shifts and long periods without medium during
washing steps. We recommend performing
transfection experiments in the presence of
serum, so that cells are not deprived of neces-
sary growth factors and nutrients.
Vector related Toxic effects may arise if a plasmid encoding a
influences toxic protein is used or if too much plasmid with
a high expression rate is used. Conversely, if
insufficient plasmid with low expression rate is
used, transfection efficiency may be too low.
When performing transfection experiments, it is
useful to perform controls. A known reporter
construct can help determine if the promoter
works in the target cell line. In addition, using a
plasmid with no insert can help determine if the
gene of interest is toxic.
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18 PolyFect Transfection Reagent Handbook 09/2000
Observation Possible Cause Comments and suggestions
Variable Inconsistent cell Count cells prior to seeding to ensure that the
transfection confluency in same number of cells is seeded for each
efficiencies in replicate experiments experiment. Keep incubation times between
replicate seeding and complex addition consistent
experiments between experiments.
Possible mycoplasma Mycoplasma contamination influences transfection
contamination efficiency. Variations in the growth behavior of
mycoplasma-infected cells will lead to different
transfection efficiencies between replicate
experiments.
Cells have been Cells that have been passaged for an extended
passaged too many number of times tend to change their growth
times behavior, morphology and transfectability.
When cells with high passage numbers are
used for replicate experiments, decreased trans-
fection efficiencies may be observed in later
experiments. We recommend using cells with
low passage number (<50 splitting cycles).
Serum variability Variations in serum quality can lead to variations
in transfection efficiency. In general, it is advisable
to test a small lot of serum from a reputable
supplier with a control cell line before performing
transfection experiments. Once a given lot has
yielded satisfactory and reproducible results,
additional sera from the same lot should be
purchased.
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PolyFect Transfection Reagent Handbook 09/2000 19
Appendix A: Composition of Buffers
Buffer Composition Storage
1x PBS 137 mM NaCl Room temperature
(phosphate buffered saline) 2.7 mM KCl
4.3 mM Na
2
HPO
4
1.47 mM KH
2
PO
4
Adjust to a final pH of 7.4
1x TE buffer, pH 7 to pH 8 10 mM Tris·Cl Room temperature
1 mM EDTA
Adjust to a pH between 7 and 8
Appendix B: Background Information
Transfection Principle
Transfection — delivery of foreign molecules such as DNA into eukaryotic cells — has
become a powerful tool for the study and control of gene expression, for example in
biochemical characterization, mutational analyses, or investigation of the effects of
regulatory elements or cell growth behavior. Two principally different transfection
techniques can be used; transient transfection and stable transfection. For further back-
ground information on transfection, please refer to current molecular biology manuals (1, 2).
Transient transfection
When cells are transiently transfected, the DNA is introduced into the nucleus of the cell,
but does not integrate into the chromosome. This means that many copies of the gene of
interest are present, leading to high levels of expressed protein. Transcription of the trans-
fected gene can be analyzed within 24 to 96 hours after introduction of the DNA depending
on the construct used. Transient transfection is most efficient when supercoiled plasmid
DNA is used.
Stable transfection
With stable or permanent transfection, the transfected DNA is either integrated into the
chromosomal DNA or maintained as an episome. Stable transfection involving integration
of the DNA is most efficient when linearized plasmid DNA is used, since linearization
facilitates recombination of the DNA with the host cell chromosome. Cells which have
successfully integrated the DNA of interest or have maintained episomal plasmid DNA
can be distinguished by using selectable markers. Frequently used selectable markers are
the genes encoding aminoglycoside phosphotransferase (APH; neo
R
gene) or hygromycin
B phosphotransferase (HPH). Other selectable markers are the genes encoding adenosine
deaminase (ADA), dihydrofolate reductase (DHFR), thymidine kinase (TK) or xanthine-
guanine phosphoribosyl transferase (XGPRT; gpt gene).
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PolyFect Transfection Reagent Handbook 09/200020
Primary cells and cell lines
Depending on their origin, cell cultures or cell lines grow as an adherent monolayer or in
suspension. Cells or cell lines vary greatly with respect to their growth behavior and
nutritional requirements (1). Optimization of the cell culture technique is therefore necessary
to ensure that cells are healthy and in optimal condition for transfection. For extensive infor-
mation on culturing of cells, please refer to the manual Culture of Animal Cells (1).
Adherent cells
Adherent cells are anchorage-dependent and propagate as a monolayer attached to the
culture vessel. This attachment is essential for proliferation. Most cells derived from tissues are
anchorage-dependent with the exception of hemopoetic cells (cells derived from blood).
Suspension cells
Suspension cells are able to survive and proliferate without attachment. Hemopoetic cells,
transformed cell lines, and cells from malignant tumors can be grown in suspension.
Primary cell cultures
Primary cell cultures arise from the outgrowth of migrating cells from a piece of tissue or
by enzymatic, chemical, or mechanical dispersal of the tissue. Primary cell cultures are
morphologically most similar to the parent tissue.
Finite cell lines
Finite cell lines are formed after the first subculturing (passaging) of a primary cell culture,
and can be propagated and subcultured several times.
Continuous cell lines
There is a limit to the number of generations that a finite cell line can be propagated. After
that it will either die out or acquire a stable, heritable alteration, giving rise to a continuous
cell line. This alteration is commonly known as in vitro transformation or immortalization,
and frequently correlates with tumorigenicity.
Transfection considerations
Media and supplements
Media are composed of a mixture of essential salts, nutrients, and buffering agents. Sterile
media are usually purchased in solution. Alternatively, packaged premixed powders are
available. Most media purchased are guaranteed to be mycoplasma- and endotoxin-free.
Supplements to the media must include glutamine and can include nonessential amino
acids, sodium pyruvate, and antibiotics. Some common media include DMEM, F12,
DMEM/F12, RPMI 1640, MEM, and S-MEM.
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PolyFect Transfection Reagent Handbook 09/2000 21
Serum
In most cases media are supplemented shortly before use with serum. Fetal calf serum
(FCS) is often used, but for some applications less expensive sera like horse- or calf serum
can be used. Generally serum is a partially undefined material, which contains growth-
and attachment factors and may show considerable variation in the ability to support
growth of particular cells. Variations in the serum quality can also lead to variation in trans-
fection efficiency. In general, it is advisable to test a small lot of serum from a reputable
supplier with a control cell line and assay before performing transfection experiments.
Once a given lot has been shown to yield satisfactory and reproducible results, additional
sera from the same lot should be purchased.
Transfection methods
Of the variety of different transfection methods described in literature (2, 3), the DEAE-dextran
method, the calcium-phosphate method, electroporation, and liposome-mediated trans-
fection are the most commonly used. Each individual method has its characteristic advantages
and disadvantages and the choice of transfection method strongly influences transfection
results. PolyFect Transfection Reagent represents a transfection reagent based on activated-
dendrimer technology, and has been designed to offer very high transfection efficiencies,
good reproducibility, and reduced cytotoxic effects. In addition, optimized protocols for
commonly used cell lines minimize optimization time.
Plasmid DNA quality
The quality of the plasmid DNA strongly influences the results of transfection experiments.
Therefore only plasmid DNA of the highest quality, which is completely free of contami-
nating RNA, genomic DNA, and proteins, should be used. DNA purified with QIAGEN,
QIAfilter and HiSpeed Plasmid Kits is ideally suited for transfection of most cell lines. For
transfection of endotoxin sensitive cells, we recommend using DNA purified with the
EndoFree Plasmid Kit. This kit efficiently removes bacterial lipopolysaccharide molecules
during the plasmid purification procedure, ensuring optimal transfection results.
Genetic Reporter Systems
After cloning a gene of interest, transfection is a useful tool to determine how cis-acting
sequences, such as promoters and enhancers, and trans-acting factors, such as transcription
factors, act together to control eukaryotic gene expression. Common methods to monitor
gene expression involve using techniques such as northern blot analysis or nuclease
protection assays to quantitate the specific mRNAs transcribed from the gene of interest.
Since these procedures are time-consuming and inconvenient for multiple samples (resulting
from multiple constructs), an alternative approach is to link the presumed cis-acting
sequences from the gene of interest to the coding sequence of an unrelated reporter gene
(see examples below) (2, 3). The reporter gene provides an indirect way of measuring
how such regulatory sequences influence gene expression. Reporter genes are also useful
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PolyFect Transfection Reagent Handbook 09/200022
in serving as controls. Transfection efficiencies between transfection experiments can be
standardized by comparing the expression of the reporter gene product. Further
information on genetic reporter systems can be obtained from current molecular biology
manuals (2, 3). In choosing a suitable reporter system, several considerations should be
taken into account. First, the reporter gene should be absent from the cells used in the study
or easily distinguished from the native form of the gene. Second, the assay for the reporter
gene product should be quick, easy, sensitive, and inexpensive. In particular, a broad
linear range is important to enable detection of both small and large changes in reporter
gene expression. Finally, the presence of the reporter gene should not affect the physiology
of the cells being used.
Chloramphenicol acetyltransferase
The prokaryotic enzyme chloramphenicol acetyltransferase (CAT) is commonly used as a
reporter. This enzyme catalyzes the transfer of acetyl groups from acetyl-coenzyme A to
chloramphenicol. In the common CAT assay, cell lysates prepared from transfected cells
are incubated with
14
C-labeled chloramphenicol. The resulting acetylated and unacetylated
forms of chloramphenicol are separated by thin-layer chromatography. A qualitative
estimate of CAT activity can be obtained simply by exposing the plates to X-ray film. For
quantitative analysis, the separated bands can be scraped from the thin-layer plate and
the levels of radioactivity measured in a scintillation counter. Currently, a CAT ELISA is
also often used. In this assay the total expression of the chloramphenicol acetyltransferase
is measured via antibody detection, in contrast to the classic CAT assay described above,
which determines only the active protein.
Firefly luciferase
Luciferase catalyses a bioluminescent reaction involving the substrate luciferin, ATP, Mg
2+
,
and molecular oxygen. When these components are mixed with cell lysates containing
luciferase, a flash of light is emitted. Light signals are detected using a luminometer or a
liquid scintillation counter.
β-Galactosidase
The prokaryotic enzyme β-galactosidase can be assayed colorimetrically using the
substrate o-nitrophenyl-β-
D-galactopyranoside (ONPG). The hydrolysis of ONPG by
β-galactosidase yields a yellow-colored product, o-nitrophenol, which can be measured
photometrically.
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PolyFect Transfection Reagent Handbook 09/2000 23
Human growth hormone (hGH)
The assay for human growth hormone is based on immunological detection of hGH secreted
by transfected cells. Specific
125
I-labeled antibodies against hGH are used and results are
monitored in a scintillation counter. Currently, a sandwich-ELISA is also often used, which
involves an antibody coupled ELISA plate. The hGH protein binds to the antibody on the
plate, a digoxygenated antibody binds to hGH, and a secondary antibody coupled to
alkaline phosphatase is used for detection.
Green fluorescent protein
Green fluorescent protein (GFP), originally isolated from the jellyfish Aequorea victoria (2),
has the ability to absorb blue light and emit green light. This unique protein can be
expressed in mammalian cells and protein expression can be visually monitored in living
cells. Although the system provides a convenient way to detect protein expression without a
specific assay, quantitative analysis is limited. This reporter gene system is best suited for
in situ detection of gene expression, such as localization studies of fusion proteins within cells.
References
1. Freshney, R.I., Culture of Animal Cells, A Manual of Basic Technique, 3rd Ed.,
Wiley-Liss.
2. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D. D., Seidman, J. G., Smith, J. A.,
and Struhl, K., eds. (1991) Current Protocols in Molecular Biology, New York: Wiley
Interscience.
3. Sambrook, J., Fritsch, E.F., and Maniatis, T., eds. (1989) Molecular cloning — a
laboratory manual, 2nd Ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
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PolyFect Transfection Reagent Handbook 09/200024
Product Use Limitations
PolyFect Transfection Reagent is developed, designed, and sold for research purposes
only. It is not to be used for human diagnostic or drug purposes or to be administered to
humans unless expressly cleared for that purpose by the Food and Drug Administration in
the USA or the appropriate regulatory authorities in the country of use. All due care and
attention should be exercised in the handling of many of the materials described in this text.
Product Warranty and Satisfaction Guarantee
QIAGEN guarantees the performance of all products in the manner described in our product
literature. The purchaser must determine the suitability of the product for its particular use.
Should any product fail to perform satisfactorily due to any reason other than misuse,
QIAGEN will replace it free of charge or refund the purchase price. We reserve the right
to change, alter, or modify any product to enhance its performance and design. If a
QIAGEN product does not meet your expectations, simply call your local Technical
Service Department or distributor. We will credit your account or exchange the product
— as you wish.
A copy of QIAGEN terms and conditions can be obtained on request, and is also provided
on the back of our invoices. If you have questions about product specifications or
performance, please call QIAGEN Technical Services or your local distributor listed on
the last page.
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PolyFect Transfection Reagent Handbook 09/2000 25
Ordering Information
Product Contents Cat. No.
PolyFect Transfection Reagent for optimized transfection of COS-7,
NIH/3T3, HeLa, 293, and CHO cells
PolyFect Transfection Reagent For 25–65 transfections in 301105
(1 ml) 60 mm dishes or 50–100 transfections
in 6-well plates
PolyFect Transfection Reagent For 100–260 transfections in 301107
(4 x 1 ml) 60 mm dishes or
200–400 transfections
in 6-well plates
PolyFect Transfection Reagent For 2500–6500 transfections in 301108
(100 ml) 60 mm dishes or
5000–10,000 transfections
in 6-well plates
SuperFect Transfection Reagent, based on activated-dendrimer technology
SuperFect Transfection Reagent For 40 transfections in 60 mm dishes 301305
(1.2 ml) or 160 transfections in 12-well plates
SuperFect Transfection Reagent For 160 transfections in 60 mm dishes 301307
(4 x 1.2 ml) or 640 transfections in 12-well plates
Effectene Transfection Reagent, the next generation in lipid technology
Effectene Transfection Reagent For 40 transfections in 60 mm dishes 301425
(1 ml) or 160 transfections in12-well plates
Effectene Transfection Reagent For 160 transfections in 60 mm dishes 301427
(4 x 1 ml) or 640 transfections in 12-well plates
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PolyFect Transfection Reagent Handbook 09/200026
Ordering Information
Product Contents Cat. No.
Transfection-related products*
EndoFree Plasmid Kits
EndoFree Plasmid Maxi Kit (10) 10 QIAGEN-tip 500, Reagents, 12362
10 QIAfilter Maxi Cartridges,
Endotoxin-free Buffers
EndoFree Plasmid Mega Kit (5) 5 QIAGEN-tip 2500, Reagents, 12381
5 QIAfilter Mega-Giga Cartridges,
Endotoxin-free Buffers
EndoFree Plasmid Giga Kit (5) 5 QIAGEN-tip 10000, Reagents, 12391
5 QIAfilter Mega-Giga Cartridges,
Endotoxin-free Buffers
HiSpeed Plasmid Kit
HiSpeed Plasmid Midi Kit (25) 25 HiSpeed Midi Tips, 12643
25 QIAfilter Midi Cartridges,
25 QIAprecipitator Midi Modules,
plus syringes, reagents, buffers
QIAfilter Plasmid Kits
QIAfilter Plasmid Midi Kit (25) 25 QIAGEN-tip 100, Reagents, Buffers, 12243
25 QIAfilter Midi Cartridges
QIAfilter Plasmid Midi Kit (100) 100 QIAGEN-tip 100, Reagents, 12245
Buffers, 100 QIAfilter Midi Cartridges
QIAfilter Plasmid Maxi Kit (10) 10 QIAGEN-tip 500, Reagents, Buffers, 12262
10 QIAfilter Maxi Cartridges
QIAfilter Plasmid Maxi Kit (25) 25 QIAGEN-tip 500, Reagents, Buffers, 12263
25 QIAfilter Maxi Cartridges
QIAfilter Plasmid Mega Kit (5) 5 QIAGEN-tip 2500, Reagents, Buffers, 12281
5 QIAfilter Mega-Giga Cartridges
QIAfilter Plasmid Giga Kit (5) 5 QIAGEN-tip 10000, Reagents, Buffers, 12291
5 QIAfilter Mega-Giga Cartridges
* Other prep and kit sizes available
1015222HBPolyFect_0900WW.qxd 28.08.2000 17:03 Uhr Seite 26
QIAGEN Companies
Please see the inside front cover for contact information for your local QIAGEN office.
QIAGEN Distributors
Argentina
Tecnolab S.A.
Charlone 144 - 1427
Capital Federal
Tel: (011) 4555 0010
Fax: (011) 4553 3331
E-mail: [email protected]
Web site: www.tecnolab.com.ar
Austria/Hungary/Slovenia
R. u. P. MARGARITELLA
Ges. m.b.H.
BIOTRADE
Breitenfurter Straße 480
1230 Wien-Rodaun
Austria
Tel: (01) 889 18 19
Fax: (01) 889 18 19 20
E-mail: [email protected]
Belgium/Luxemburg
Westburg b.v.
P.O. Box 214
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The Netherlands
Tel: 0800-1-9815
Fax: (31) 33-4951222
E-mail: [email protected]
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or 0800551321
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Tel: (305) 828-3818
Fax: (305) 828-3819
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E-mail: [email protected]
Web site: www.merckeurolab.dk
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Fax: 525 7210
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Tel: (09)-804 551
Fax: (09)-804 55200
E-mail: [email protected]
Web site: www.merckeurolab.fi
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Fax: (01)-646 27 48
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or (011)-515 9346
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or 1-800 20 22 20 (toll free)
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Web site: www.westburg.co.il
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LRS Laboratories, Inc.
SongBuk P.O. Box 61
Seoul, 136-600
Tel: (02) 924-86 97
Fax: (02) 924-86 96
E-mail:[email protected]
Malaysia
Research Biolabs Sdn. Bhd.
79A Jalan SS15/4C
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Tel: (09)980 6700
or 0800933966
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Tel: 22 90 00 00
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Web site: www.merckeurolab.no
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Tel: (071) 351 41 06
or 0601 70 60 07
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E-mail: [email protected]
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Portugal
IZASA PORTUGAL, LDA
Rua do Proletariado, 1 - Quinta do
Paizinho
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Tel: (1)-424 73 64
Fax: (1)-417 26 59
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Tel: 445 7927
Fax: 448 3966
E-mail:
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BIO-CONSULT Slovakia spol. s.r.o.
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P.O. Box 23681
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IZASA, S.A.
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Tel: (93) 902.20.30.90
Fax: (93) 902.22.33.66
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Merck Eurolab AB
Fagerstagatan 18A
16394 Sp
°
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Fax: (08) 760 45 20
E-mail: [email protected]
Web site: www.merckeurolab.se
Taiwan
TAIGEN Bioscience Corporation
3F. No. 306, Section 4
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Taiwan, R.O.C.
Tel: (02) 2880 2913
Fax: (02) 2880 2916
E-mail: [email protected]
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(Charan 13) Bangkokyai
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QIAGEN Importers
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Abdulla Fouad Co. Ltd.
Medical Supplies Division
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Tel: (03) 8324400
Fax: (03) 8346174
E-mail:
All other countries
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