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Protocols for X-tremeGENE transfecting.
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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
X-tremeGENE 9 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
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
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
8Commonly Used Cell Types
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.
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
9Commonly Used Cell Types
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.
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
10Commonly Used Cell Types
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.
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
11Commonly Used Cell Types
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.
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
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.
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
13Difficult-to-Transfect Cells
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.
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
14Difficult-to-Transfect Cells
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.
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
15Difficult-to-Transfect Cells
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.
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
16Difficult-to-Transfect Cells
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.
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
17Difficult-to-Transfect Cells
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.
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
18Difficult-to-Transfect Cells
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.
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
19Difficult-to-Transfect Cells
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.
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 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
Please be aware that the protocols are suggestions and that optimal conditions must be determined empirically.
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.
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
21Primary Cells and Stem Cells
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.
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
22Primary Cells and Stem Cells
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.
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
23Primary Cells and Stem Cells
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.
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
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.
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
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.
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.
26General QuickProtocols
X-tremeGENE 9 DNA Transfection Reagent
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.
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 : 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
6 : 1X-tremeGENE 9 DNA Transfection Reagent 6 l 30 l
DNA 1 g 5 g
6 : 2X-tremeGENE 9 DNA Transfection Reagent 6 l 30 l
DNA 2 g 10 g
9
DNA
27General QuickProtocols
X-tremeGENE HP DNA Transfection Reagent
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.
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 : 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
2 : 1X-tremeGENE HP DNA Transfection Reagent 2 l 10 l
DNA 1 g 5 g
3 : 1X-tremeGENE HP DNA Transfection Reagent 3 l 15 l
DNA 1 g 5 g
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