x-tremegene protocols

X-tremeGENE ProtocolsEasy DNA Transfection

Dear Transfection Customer,

lab time is valuable, especially if you need to establish cell culture experiments. Roches cell-type specific X-tremeGENE protocols, available at your fingertips at the right time and in an easily accessible format can help you establish your transfection experiments more efficiently.We hope you find 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 4Selection Table 6Ordering 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

4X-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. Efficiency 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.

Nor

mal

ized

Tra

nsfe

ctio

n Ef

ficie

ncy

HT-1080 RAW 264.7 K-562PC-3

15

10

5

0

Reference ReagentX-tremeGENE HP DNA Transfection ReagentCompetitor LTX P

5X-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

oxic

ity

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

6Note: 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


Transfection of DNA +++ +++

Efficiency with standard cell lines (e.g., COS-7, HEK-293, HeLa, NIH 3T3, CHO-K1)

+++ +++

Efficiency with difficult-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


06 366 244 001 06 366 236 001 06 366 546 001

0.4 ml 1.0 ml 5 x 1 ml


06 365 779 001 06 365 787 001 06 365 809 001

0.4 ml 1.0 ml 5 x 1 ml

Ordering Information

7Commonly Used Cell Types

CHO-K1 Cells

Plate CHO-K1 cells at a density of 1.5 104 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


COS-7 Cells

Plate COS-7 cells at a density of 8.0 9.0 103 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.


Place 200 l diluent in a sterile tube. Add 6 l X-tremeGENE 9 DNA Transfection Reagent to the

diluent. Pipet gently to mix.






Experimental result: COS-7 cells were successfully transfected with pcDNA3.1-GFP plasmid using X-tremeGENE 9 DNA Transfection Reagent.


9

DNA

Transfection of


HeLa Cells

Plate HeLa cells at a density of 1.5 104 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.






Experimental result: HeLa cells were successfully transfected with pcDNA3.1-GFP plasmid using X-tremeGENE 9 DNA Transfection Reagent.


DNA

9

Transfection of


NIH-3T3 Cells

Plate NIH-3T3 cells at a density of 1.5 104 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.









Experimental result: NIH-3T3 cells were successfully trans-fected with pcDNA3.1-GFP plasmid using X-tremeGENE 9 DNA Transfection Reagent.

9

DNA


Transfection of


HEK-293 Cells

Plate HEK-293 cells at a density of 3.0 3.5 104 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.





Experimental result: HEK-293 cells were successfully trans-fected with pcDNA3.1-GFP plasmid using X-tremeGENE HP DNA Transfection Reagent.

HP

DNA


Transfection of

12Difficult-to-Transfect Cells

PC-3 Cells

Plate PC-3 cells at a density of 1.0 1.2 104 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.








Experimental result: PC-3 cells were successfully transfected with pcDNA3.1-GFP plasmid using X-tremeGENE HP DNA Transfection Reagent.

HP

DNA


Transfection of


HCT 116 Cells

Plate HCT 116 cells at a density of 3.0 4.0 105 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.





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


Transfection of


A549 Cells

Plate A549 cells at a density of 2.8 104 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.








HP

DNA


Transfection of


MCF-7 Cells

Plate MCF-7 cells at a density of 1.2 1.8 104 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.









Experimental result: MCF-7 cells were successfully trans-fected with pcDNA3.1-GFP plasmid using X-tremeGENE 9 DNA Transfection Reagent.

9

DNA


Transfection of


HuH-7 Cells

Plate HuH-7 cells at a density of 1.0 105 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.


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.


One hour before adding the complexes to cells, refresh the medium with 1.5 ml of Opti-MEM I Reduced Serum Medium.



The next day refresh the medium with complete growth medium. Incubate cells for 24 72 hours before measuring protein

expression.

HP

DNA


Transfection of


U-2 OS Cells

Plate U-2 OS cells at a density of 2.5 3.0 104 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.








Experimental result: U-2 OS cells were successfully trans-fected with pcDNA3.1-GFP plasmid using X-tremeGENE HP DNA Transfection Reagent.

HP

DNA


Transfection of


HepG2 Cells

Plate HepG2 cells at a density of 2.0 2.5 104 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.









Experimental result: HepG2 cells were successfully trans-fected with pcDNA3.1-GFP plasmid using X-tremeGENE 9 DNA Transfection Reagent.

9

DNA


Transfection of


Neuro-2a Cells

Protocol provided by a customer.Plate Neuro-2a cells at a density of 1.0 105 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.









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 defi ning the criteria for the performance of the specifi c 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


Transfection of

20Primary Cells and Stem Cells

Primary MEF Cells

Plate primary MEF cells at a density of 0.5 1.0 105 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.







expression.

HP

DNA


Transfection of


Human Primary Fibroblasts from Pre-Skin

Plate human primary fibroblast cells at a density of 1.0 105 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% con uency). Incubate cell cultures overnight.








Experimental result: Human primary fibroblasts were successfully transfected with GFP-encoding plasmid using X-tremeGENE HP DNA Transfection Reagent.

HP

DNA


Transfection of


Murine Mesenchymal Stem Cells EK8

Plate EK8 cells at a density of 3.0 104 cells/well. Plate cells (3 105 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.








HP

DNA


Transfection of


Human Mesenchymal Stem Cells

Plate hMSCs at a density of 8.0 104 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.








Experimental result: human mesenchymal stem cells were successfully transfected with GFP-encoding plasmid using X-tremeGENE HP DNA Transfection Reagent.

HP

DNA


Transfection of

24Packaging Cells and Insect Cells

HEK-293T Cells for Lentiviral Production

Plate HEK-293T cells at a density of 5.0 105 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.


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.






Experimental result: HEK-293T cells were successfully trans-fected with pGIPZ-eGFP plasmid using X-tremeGENE HP DNA Transfection Reagent.

HP

DNA


Transfection of

25Packaging Cells and Insect Cells

SF9 Insect Cells

Plate SF9 cells at a density of 0.5 106 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.







expression.

Transfection of

For life science research only. Not for use in diagnostic procedures.

HP

DNA


26General QuickProtocols


Cell preparation for transfectionPlate cells approx. 24 hours before transfection making sure cells are at optimal concentration (70 90 % confluency).

Steps prior to transfectionAllow X-tremeGENE 9 DNA Transfection Reagent, DNA and diluent (Opti:MEMI 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.



expression.

Volumes of X-tremeGENE 9 DNA Transfection Reagent and amounts of DNA for various ratios

Ratio Transfection Reagent : DNAMinimal transfection complex volume

Volume for a whole 96-well plate

Serum-free medium to a final volume of 100 l 500 l

3 : 1X-tremeGENE 9 DNA Transfection Reagent 3 l 15 l

DNA 1 g 5 g


DNA 1 g 5 g


DNA 2 g 10 g

9

DNA

27General QuickProtocols


Cell preparation for transfectionPlate cells approx. 24 hours before transfection making sure cells are at optimal concentration (70 90 % confluency).

Steps prior to transfectionAllow X-tremeGENE 9 DNA Transfection Reagent, DNA and diluent (Opti:MEMI 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.



expression.

HP

DNA

Volumes of X-tremeGENE 9 DNA Transfection Reagent and amounts of DNA for various ratios

Ratio Transfection Reagent : DNAMinimal transfection complex volume

Volume for a whole 96-well plate

Serum-free medium to a final volume of 100 l 500 l

1 : 1X-tremeGENE HP DNA Transfection Reagent 1 l 5 l

DNA 1 g 5 g


DNA 1 g 5 g


DNA 1 g 5 g

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