X-tremeGENE Protocols

Easy DNA Transfection

Dear Transfection Customer,
lab time is valuable, especially if you need to establish cell
culture experiments. Roches cell-type specic X-tremeGENE
protocols, available at your ngertips at the right time and in an
easily accessible format can help you establish your transfection
experiments more efciently.
We hope you nd this new compendium of protocols useful for
your laboratory work.
Thank you for using Roche X-tremeGENE DNA Transfection
Reagents, and we wish you continued success in your research
projects.
Sincerely
Roche Applied Science
Note: The protocols in this compendium are guidelines.
Successful transfections are dependent on a multitude of
parameters, including cell density, passage number of cells,
cell cycle, nature of DNA plasmid backbone, purity of the
DNA preparation, and the strength of the promoter. Therefore,
transfection conditions must be determined empirically and
optimized individually for your cell culture conditions.
For life science research only. Not for use in diagnostic procedures.
X-TREMEGENE is a trademark of Roche.
Other brands or product names are trademarks of their respective holders.
Find the Right X-tremeGENE Protocol
Advantages of X-tremeGENE Reagents 4
Selection Table 6
Ordering Information 6
CHO-K1 7
COS-7 8
HeLa 9
NIH-3T3 10
HEK-293 11
PC-3 12
HCT 116 13
A549 14
MCF-7 15
HuH-7 16
U-2 OS 17
HepG2 18
Neuro-2a 19
Primary MEF 20

Human Primary Fibroblasts from Pre-Skin 21
Murine Mesenchymal Stem Cells EK8 22
Human Mesenchymal Stem Cells 23
HEK-293T Cells for Lentiviral Production 24

SF9 Insect Cells 25
26
Commonly Used Cell Types
Difficult-to-Transfect Cells
Primary Cells and Stem Cells
Packaging Cells and Insect Cells
General QuickProtocols
4
X-tremeGENE HP DNA Transfection Reagent
High Performance
Due to its optimized formulation, X-tremeGENE HP Reagent
efficiently transfects a broad range of eukaryotic cells, including
insect cells and hard-to-transfect cell lines.
Achieve high levels of efficiency
for hard-to-transfect cell lines.
Benefit from an easy-to-use reagent,
free of animal-derived components.
Increase experimental throughput
using a simple and fast protocol.
Figure 1. X-tremeGENE HP DNA Transfection Reagent outperforms reagent
LTX P. Four hard-to-transfect cell lines were transfected in medium using
X-tremeGENE HP Reagent. Efciency was measured as a ratio of GFP transfected
cells versus whole cell number, and normalized to the reference reagent set at 1.
Error bars show the standard deviation of the mean of triplicates.
N
o
r
m
a
l
i
z
e
d

T
r
a
n
s
f
e
c
t
i
o
n

E
f

c
i
e
n
c
y
HT-1080 RAW 264.7 K-562 PC-3
15
10
5
0
Reference Reagent
X-tremeGENE HP DNA Transfection Reagent

Competitor LTX P
5
X-tremeGENE 9 DNA Transfection Reagent
High Cell Survival
Due to its low cytotoxicity, minimal optimization, and high
transfection efficiency in commonly used cell lines, such as
HeLa, CHO-K1, NIH-3T3 and COS-7, X-tremeGENE 9 Reagent is
perfectly suited for applications in all fields of cellular analysis,
even with serum.
Obtain maximum cell viability
after transfection.
Generate physiologically relevant data
using a reagent with low cytotoxic effects.
Save time and effort,
and avoid time-consuming optimization.
Figure 2. High cell survival using X-tremeGENE 9 DNA Transfection
Reagent. HeLa cells were transfected. Reagent cytotoxicity was measured using
the Roche LDH Cytotoxicity Detection Kit. The ratios of l reagent to
l DNA used, are indicated. X-tremeGENE 9 Reagent showed lower cytotoxicity,
compared to the competitor reagents.
Competitor
LTX P Reagent
%

T
o
x
i
c
i
t
y
2:1 3:1 2:1 3:1 1.5:1:1 3:1:1
40
30
20
10
0
X-tremeGENE 9 DNA
Transfection Reagent
Competitor
L2K Reagent
6
Note: Above data were produced using either a GFP-encoding
pcDNA3.1 plasmid or a Luciferase-encoding pCI plasmid, both with
the cytomegalovirus (CMV) promoter. These recommendations are
guidelines based on experimental findings.
The optimal reagent: DNA ratio must be determined empirically.
Selection Table for X-tremeGENE DNA Transfection Reagents
X-tremeGENE HP
DNA Transfection
Reagent
X-tremeGENE 9
DNA Transfection
Reagent
Transfection of DNA +++ +++
Efciency with standard cell lines
(e.g., COS-7, HEK-293, HeLa,
NIH 3T3, CHO-K1)
+++ +++
Efciency with
difcult-to-transfect cells
(e.g., HT 29, HCT 116, K-562)
+++ +
Transfection of primary cells ++(+) not recommended;
exceptions are
possible
Gentleness of the reagent ++(+) +++
Ease of use
(minimal optimization)
+++ +++
Cytotoxicity after transfection Very low Very low to
exceptionally low
Storage -15 to -25 C +2 to +8 C
Product Catalog Number Pack Size
X-tremeGENE HP
DNA Transfection
Reagent
06 366 244 001
06 366 236 001
06 366 546 001
0.4 ml
1.0 ml
5 x 1 ml
X-tremeGENE 9
DNA Transfection
Reagent
06 365 779 001
06 365 787 001
06 365 809 001
0.4 ml
1.0 ml
5 x 1 ml
Ordering Information
7 Commonly Used Cell Types
CHO-K1 Cells
Plate CHO-K1 cells at a density of 1.5 10
4
cells/well.
Plate cells in a volume of 100 l complete growth medium
per well in a 96-well plate 18 24 hours before transfection
(65 75% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE 9 DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 200 l diluent in a sterile tube.

Add 6 l X-tremeGENE 9 DNA Transfection Reagent

to the diluent. Pipet gently to mix.

Add 2 g plasmid DNA. (3:1 ratio of reagent to DNA).

Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 5 l transfection complex to the cells in a dropwise
manner.

Gently shake or swirl the wells or flasks to ensure even
distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
Experimental result: CHO-K1 cells were successfully
transfected with pcDNA3.1-GFP plasmid using using
X-tremeGENE 9 DNA Transfection Reagent.
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
9
DNA
Transfection of
8 Commonly Used Cell Types
COS-7 Cells
Plate COS-7 cells at a density of 8.0 9.0 10
3
cells/well.
Plate cells in a volume of 100 l complete growth medium
per well in a 96-well plate 18 24 hours before transfection
(75 85% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE 9 DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 200 l diluent in a sterile tube.

Add 6 l X-tremeGENE 9 DNA Transfection Reagent to the

diluent. Pipet gently to mix.

Add 2 g plasmid DNA. (3:1 ratio of reagent to DNA).

Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 5 l transfection complex to the cells in a dropwise

manner.

Gently shake or swirl the wells or flasks to ensure even

distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
Experimental result: COS-7 cells were successfully
transfected with pcDNA3.1-GFP plasmid using
X-tremeGENE 9 DNA Transfection Reagent.
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
9
DNA
Transfection of
9 Commonly Used Cell Types
HeLa Cells
Plate HeLa cells at a density of 1.5 10
4
cells/well.
Plate cells in a volume of 100 l complete growth medium per
well in a 96-well plate 18 24 hours before transfection
(70 90% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE 9 Transfection Reagent, DNA and diluent
(Opti-MEM

I Reduced Serum Medium or serum-free medium)

to warm to +15 C to +25 C, and vortex gently.

Place 200 l diluent in a sterile tube.

Add 6 l X-tremeGENE 9 DNA Transfection Reagent to the

diluent. Pipet gently to mix.

Add 2 g plasmid DNA. (3:1 ratio of reagent to DNA).

Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 5 l transfection complex to the cells in a dropwise
manner.

Gently shake or swirl the wells or flasks to ensure even
distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
Experimental result: HeLa cells were successfully transfected
with pcDNA3.1-GFP plasmid using X-tremeGENE 9 DNA
Transfection Reagent.
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
DNA
9
Transfection of
10 Commonly Used Cell Types
NIH-3T3 Cells
Plate NIH-3T3 cells at a density of 1.5 10
4
cells/well.
Plate cells in a volume of 100 l complete growth medium per
well in a 96-well plate 18 24 hours before transfection
(70 80% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE 9 DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 200 l diluent in a sterile tube.

Add 6 l X-tremeGENE 9 DNA Transfection Reagent

to the diluent. Pipet gently to mix.

Add 2 g plasmid DNA. (3:1 ratio of reagent to DNA).

Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 5 l transfection complex to the cells in a dropwise
manner.

Gently shake or swirl the wells or flasks to ensure even
distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
Experimental result: NIH-3T3 cells were successfully trans-
fected with pcDNA3.1-GFP plasmid using X-tremeGENE 9
DNA Transfection Reagent.
9
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
11 Commonly Used Cell Types
HEK-293 Cells
Plate HEK-293 cells at a density of 3.0 3.5 10
4
cells/well.
Plate cells in a volume of 100 l complete growth medium
per well in a 96-well plate 18 24 hours before transfection
(50 70% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE HP DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 200 l diluent in a sterile tube.

Add 2 g plasmid DNA. Pipet gently to mix.

Add 6 l X-tremeGENE HP DNA Transfection Reagent to the

diluted DNA. (3:1 ratio of reagent to DNA). Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 10 l transfection complex to the cells in a dropwise
manner.

Gently shake or swirl the wells or flasks to ensure even
distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
Experimental result: HEK-293 cells were successfully trans-
fected with pcDNA3.1-GFP plasmid using X-tremeGENE HP
DNA Transfection Reagent.
HP
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
12 Difficult-to-Transfect Cells
PC-3 Cells
Plate PC-3 cells at a density of 1.0 1.2 10
4
cells/well.
Plate cells in a volume of 100 l complete growth medium per
well in a 96-well plate 18 24 hours before transfection
(75 85% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE HP DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 200 l diluent in a sterile tube.

Add 2 g plasmid DNA. Pipet gently to mix.

Add 2 l X-tremeGENE HP DNA Transfection Reagent to the

diluted DNA. (1:1 ratio of reagent to DNA). Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 10 l transfection complex to the cells in a dropwise
manner.

Gently shake or swirl the wells or flasks to ensure even
distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
Experimental result: PC-3 cells were successfully transfected
with pcDNA3.1-GFP plasmid using X-tremeGENE HP DNA
Transfection Reagent.
HP
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
13 Difficult-to-Transfect Cells
HCT 116 Cells
Plate HCT 116 cells at a density of 3.0 4.0 10
5
cells/well.
Plate cells in a volume of 1 ml complete growth medium
per well in a 12-well plate 18 24 hours before transfection
(80% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE HP DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 100 l diluent in a sterile tube.

Add 1 g plasmid DNA. Pipet gently to mix.

Add 2 l X-tremeGENE HP DNA Transfection Reagent to the

diluted DNA. (2:1 ratio of reagent to DNA). Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add transfection complex to the cells in a dropwise manner.

Gently shake or swirl the wells or flasks to ensure even
distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
HP
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
14 Difficult-to-Transfect Cells
A549 Cells
Plate A549 cells at a density of 2.8 10
4
cells/well.
Plate cells in a volume of 150 l complete growth medium per
well in a 96-well plate 18 24 hours before transfection
(70% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE HP DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 500 l diluent in a sterile tube.

Add 5 g plasmid DNA. Pipet gently to mix.

Add 10 l X-tremeGENE HP DNA Transfection Reagent

to the diluted DNA. (2:1 ratio of reagent to DNA).
Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 15 l transfection complex to the cells in a dropwise
manner.

Gently shake or swirl the wells or flasks to ensure even
distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
HP
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
15 Difficult-to-Transfect Cells
MCF-7 Cells
Plate MCF-7 cells at a density of 1.2 1.8 10
4
cells/well.
Plate cells in a volume of 100 l complete growth medium
per well in a 96-well plate 18 24 hours before transfection
(70 90% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE 9 DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 200 l diluent in a sterile tube.

Add 6 l X-tremeGENE 9 DNA Transfection Reagent

to the diluent. Pipet gently to mix.

Add 2 g plasmid DNA. (3:1 ratio of reagent to DNA).

Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 10 l transfection complex to the cells in a dropwise
manner.

Gently shake or swirl the wells or flasks to ensure even
distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
Experimental result: MCF-7 cells were successfully trans-
fected with pcDNA3.1-GFP plasmid using X-tremeGENE 9
DNA Transfection Reagent.
9
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
16 Difficult-to-Transfect Cells
HuH-7 Cells
Plate HuH-7 cells at a density of 1.0 10
5
cells/well.
Plate cells in a volume of 2.5 ml complete growth medium per
well in a 6-well plate 18 24 hours before transfection.
Incubate cell cultures overnight.
Allow X-tremeGENE HP DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 100 l diluent in a sterile tube.

Add 1.5 g plasmid DNA. Pipet gently to mix.

Add 1.5 l X-tremeGENE HP DNA Transfection Reagent

to the diluted DNA. (1:1 ratio of reagent to DNA).
Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

One hour before adding the complexes to cells, refresh the

medium with 1.5 ml of Opti-MEM I Reduced Serum Medium.

Add 100 l transfection complex to the cells in a dropwise

manner.

Gently shake or swirl the wells or flasks to ensure even

distribution over the entire plate.

The next day refresh the medium with complete growth medium.

Incubate cells for 24 72 hours before measuring protein

expression.
HP
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
17 Difficult-to-Transfect Cells
U-2 OS Cells
Plate U-2 OS cells at a density of 2.5 3.0 10
4
cells/well.
Plate cells in a volume of 100 l complete growth medium
per well in a 96-well plate 18 24 hours before transfection
(70 90% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE HP DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 200 l diluent in a sterile tube.

Add 2 g plasmid DNA. Pipet gently to mix.

Add 4 l X-tremeGENE HP DNA Transfection Reagent

to the diluted DNA. (2:1 ratio of reagent to DNA).
Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 10 l transfection complex to the cells in a dropwise
manner.

Gently shake or swirl the wells or flasks to ensure even
distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
Experimental result: U-2 OS cells were successfully trans-
fected with pcDNA3.1-GFP plasmid using X-tremeGENE HP
DNA Transfection Reagent.
HP
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
18 Difficult-to-Transfect Cells
HepG2 Cells
Plate HepG2 cells at a density of 2.0 2.5 10
4
cells/well.
Plate cells in a volume of 100 l complete growth medium
per well in a 96-well plate 18 24 hours before transfection
(60 70% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE 9 DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 500 l diluent in a sterile tube.

Add 30 l X-tremeGENE 9 DNA Transfection Reagent

to the diluent. Pipet gently to mix.

Add 10 g plasmid DNA. (3:1 ratio of reagent to DNA).

Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 5 l transfection complex to the cells in a dropwise

manner.

Gently shake or swirl the wells or flasks to ensure even

distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
Experimental result: HepG2 cells were successfully trans-
fected with pcDNA3.1-GFP plasmid using X-tremeGENE 9
DNA Transfection Reagent.
9
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
19 Difficult-to-Transfect Cells
Neuro-2a Cells
Protocol provided by a customer.
Plate Neuro-2a cells at a density of 1.0 10
5
cells/well.
Plate cells in a volume of 500 l complete growth medium
per well in a 24-well plate 24 hours before transfection
(30 40% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE 9 DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 100 l diluent in a sterile tube.

Add 8 l X-tremeGENE 9 DNA Transfection Reagent

to the diluent. Pipet gently to mix.

Add 2 g plasmid DNA. (4:1 ratio of reagent to DNA).

Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 100 l transfection complex to the cells in a dropwise

manner.

Gently shake or swirl the wells or flasks to ensure even

distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
Note: Data and experimental conditions included in Protocols provided by
customers are the sole responsibility of the customers that have submitted them.
Roche was neither involved in establishing the experimental conditions nor in
dening the criteria for the performance of the specic assays. Roche therefore
cannot take any responsibility for performance or interpretation of results
obtained for the biological target parameter(s) described by the authors or other
users using a similar experimental approach.expression.
Experimental result: Neuro-2a cells were successfully trans-
fected with CMV6-GFP plasmid using X-tremeGENE 9 DNA
Transfection Reagent.
9
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
20 Primary Cells and Stem Cells
Primary MEF Cells
Plate primary MEF cells at a density of 0.5 1.0 10
5
cells/well.
Plate cells in a volume of 1 ml complete growth medium
per well in a 12-well plate 18 24 hours before transfection
(60% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE HP DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 100 l diluent in a sterile tube.

Add 1 g plasmid DNA. Pipet gently to mix.

Add 4 l X-tremeGENE HP DNA Transfection Reagent

to the diluted DNA. (4:1 ratio of reagent to DNA).
Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add transfection complex to the cells in a dropwise manner.

Gently shake or swirl the wells or flasks to ensure even

distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
HP
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
21 Primary Cells and Stem Cells
Human Primary Fibroblasts
from Pre-Skin
Plate human primary fibroblast cells at a density of
1.0 10
5
cells/well. Plate cells in a volume of 2 ml complete
growth medium per well in a 6-well plate 18 24 hours
before transfection (40 50% conuency). Incubate cell cultures
overnight.
Allow X-tremeGENE HP DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 200 l diluent in a sterile tube.

Add 2 g plasmid DNA. Pipet gently to mix.

Add 6 l X-tremeGENE HP DNA Transfection Reagent

to the diluted DNA. (3:1 ratio of reagent to DNA).
Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 200 l transfection complex to the cells in a dropwise

manner.

Gently shake or swirl the wells or flasks to ensure even

distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
Experimental result: Human primary fibroblasts were
successfully transfected with GFP-encoding plasmid using
X-tremeGENE HP DNA Transfection Reagent.
HP
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
22 Primary Cells and Stem Cells
Murine Mesenchymal Stem Cells EK8
Plate EK8 cells at a density of 3.0 10
4
cells/well. Plate cells
(3 10
5
cells/ml) in a volume of 100 l complete growth medium
per well in a 96-well plate 24 hours before transfection
(50% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE HP DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 392 l diluent in a sterile tube.

Add 8 g plasmid DNA. Pipet gently to mix.

Add 8 l X-tremeGENE HP DNA Transfection Reagent

to the diluted DNA. (1:1 ratio of reagent to DNA).
Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 10 l transfection complex to the cells in a dropwise

manner.

Gently shake or swirl the wells or flasks to ensure even

distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
HP
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
23 Primary Cells and Stem Cells
Human Mesenchymal Stem Cells
Plate hMSCs at a density of 8.0 10
4
cells/well.
Plate cells in a volume of 2 ml complete growth medium per
well in a 6-well plate 18 24 hours before transfection
(50% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE HP DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 200 l diluent in a sterile tube.

Add 2 g plasmid DNA. Pipet gently to mix.

Add 6 l X-tremeGENE HP DNA Transfection Reagent

to the diluted DNA. (3:1 ratio of reagent to DNA).
Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 200 l transfection complex to the cells in a dropwise

manner
.

Gently shake or swirl the wells or flasks to ensure even

distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
Experimental result: human mesenchymal stem cells were
successfully transfected with GFP-encoding plasmid using
X-tremeGENE HP DNA Transfection Reagent.
HP
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
24 Packaging Cells and Insect Cells
HEK-293T Cells for
Lentiviral Production
Plate HEK-293T cells at a density of 5.0 10
5
cells/well.
Plate cells in a volume of 2 ml complete growth medium
per well in a 6-well plate 18 24 hours before transfection
(60 80% confluency). Incubate cell cultures overnight.
Allow X-tremeGENE HP DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 180 l diluent in a sterile tube.

Add 2 g plasmid DNA. (20 l of a plasmid DNA mixture in

a molar ratio of 1: 2: 2 = library plasmid : packaging plasmid :
envelope plasmid). Pipet gently to mix.

Add 6 l X-tremeGENE HP DNA Transfection Reagent

to the diluted DNA. (3:1 ratio of reagent to DNA).
Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add 200 l transfection complex to the cells in a dropwise

manner.

Gently shake or swirl the wells or flasks to ensure even

distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
Experimental result: HEK-293T cells were successfully trans-
fected with pGIPZ-eGFP plasmid using X-tremeGENE HP
DNA Transfection Reagent.
HP
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
Transfection of
25 Packaging Cells and Insect Cells
SF9 Insect Cells
Plate SF9 cells at a density of 0.5 10
6
cells/well.
Plate cells in a volume of 1 ml complete growth medium per
well in a 12-well plate immediately before transfection.
Incubate cell cultures overnight.
Allow X-tremeGENE HP DNA Transfection Reagent, DNA and
diluent (Opti-MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place 100 l diluent in a sterile tube.

Add 1 g plasmid DNA. Pipet gently to mix.

Add 8 l X-tremeGENE HP DNA Transfection Reagent

to the diluted DNA. (8:1 ratio of reagent to DNA).
Pipet gently to mix.

Incubate for 15 30 min at +15 C to +25 C.

Add transfection complex to the cells in a dropwise manner.

Gently shake or swirl the wells or flasks to ensure even

distribution over the entire plate.

Incubate cells for 24 72 hours before measuring protein

expression.
Transfection of
For life science research only. Not for use in diagnostic procedures.
HP
DNA
Please be aware
that the protocols are suggestions and that optimal conditions must be determined empirically.
26 General QuickProtocols
X-tremeGENE 9
DNA Transfection Reagent
Cell preparation for transfection
Plate cells approx. 24 hours before transfection making sure
cells are at optimal concentration (70 90 % confluency).
Steps prior to transfection
Allow X-tremeGENE 9 DNA Transfection Reagent, DNA and
diluent (Opti:MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place diluent in a sterile tube.

Add X-tremeGENE 9 DNA Transfection Reagent

to the diluent.

Add plasmid DNA. Pipet gently to mix.

Incubate for 15 min at +15 C to +25 C.

Add transfection complex to the cells in a dropwise manner.

Gently shake or swirl the wells or flasks to ensure even

distribution over the entire plate.

Incubate cells for 18 72 hours before measuring protein

expression.
Volumes of X-tremeGENE 9 DNA Transfection Reagent and amounts of DNA for various ratios
Ratio Transfection Reagent : DNA
Minimal transfection
complex volume
Volume for a whole
96-well plate
Serum-free medium to a final volume of 100 l 500 l
3 : 1
X-tremeGENE 9 DNA Transfection Reagent 3 l 15 l
DNA 1 g 5 g
6 : 1
X-tremeGENE 9 DNA Transfection Reagent 6 l 30 l
DNA 1 g 5 g
6 : 2
X-tremeGENE 9 DNA Transfection Reagent 6 l 30 l
DNA 2 g 10 g
9
DNA
27 General QuickProtocols
X-tremeGENE HP
DNA Transfection Reagent
Cell preparation for transfection
Plate cells approx. 24 hours before transfection making sure
cells are at optimal concentration (70 90 % confluency).
Steps prior to transfection
Allow X-tremeGENE 9 DNA Transfection Reagent, DNA and
diluent (Opti:MEM

I Reduced Serum Medium or serum-free

medium) to warm to +15 C to +25 C, and vortex gently.

Place diluent in a sterile tube.

Add plasmid DNA. Pipet gently to mix.

Add X-tremeGENE HP DNA Transfection Reagent

to the diluted DNA.

Incubate for 15 min at +15 C to +25 C.

Add transfection complex to the cells in a dropwise manner.

Gently shake or swirl the wells or flasks to ensure even

distribution over the entire plate.

Incubate cells for 18 72 hours before measuring protein

expression.
HP
DNA
Volumes of X-tremeGENE 9 DNA Transfection Reagent and amounts of DNA for various ratios
Ratio Transfection Reagent : DNA
Minimal transfection
complex volume
Volume for a whole
96-well plate
Serum-free medium to a final volume of 100 l 500 l
1 : 1
X-tremeGENE HP DNA Transfection Reagent 1 l 5 l
DNA 1 g 5 g
2 : 1
X-tremeGENE HP DNA Transfection Reagent 2 l 10 l
DNA 1 g 5 g
3 : 1
X-tremeGENE HP DNA Transfection Reagent 3 l 15 l
DNA 1 g 5 g
Published by
Roche Diagnostics GmbH
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68305 Mannheim
Germany
2013 Roche Diagnostics.
All rights reserved.
x-tremegene.roche.com
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