X-tremeGENE Protocols Easy DNA Transfection
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1 X-tremeGENE Protocols Easy Transfection
2 Dear Transfection Customer, lab time is valuable, especially if you need to establish cell culture experiments. Roche s 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 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 plasmid backbone, purity of the 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.
3 Find the Right X-tremeGENE Protocol Advantages of X-tremeGENE Reagents 4 Selection Table 6 Ordering Information 6 Commonly Used Cell Types Difficult-to-Transfect Cells Primary Cells and Stem Cells Packaging Cells and Insect Cells General QuickProtocols CHO-K1 7 COS-7 8 HeLa 9 NIH-3T3 10 HEK PC-3 12 HCT A 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
4 4 X-tremeGENE HP 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. Normalized Transfection Efficiency Reference Reagent X-tremeGENE HP Transfection Reagent Competitor LTX P PC-3 HT-1080 RAW K-562 Figure 1. X-tremeGENE HP 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.
5 5 X-tremeGENE 9 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. 40 % Toxicity :1 3:1 2:1 3:1 1.5:1:1 3:1:1 X-tremeGENE 9 Transfection Reagent Competitor L2K Reagent Competitor LTX P Reagent Figure 2. High cell survival using X-tremeGENE 9 Transfection Reagent. HeLa cells were transfected. Reagent cytotoxicity was measured using the Roche LDH Cytotoxicity Detection Kit. The ratios of μl reagent to µl used, are indicated. X-tremeGENE 9 Reagent showed lower cytotoxicity, compared to the competitor reagents.
6 6 Selection Table for X-tremeGENE Transfection Reagents X-tremeGENE HP Transfection Reagent X-tremeGENE 9 Transfection Reagent 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) 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 Note: Above data were produced using either a GFP-encoding pc3.1 plasmid or a Luciferase-encoding pci plasmid, both with the cytomegalovirus (CMV) promoter. These recommendations are guidelines based on experimental findings. The optimal reagent: ratio must be determined empirically. Ordering Information Product Catalog Number Pack Size X-tremeGENE HP Transfection Reagent X-tremeGENE 9 Transfection Reagent ml 1.0 ml 5 x 1 ml 0.4 ml 1.0 ml 5 x 1 ml
7 Commonly Used Cell Types 7 CHO-K1 Cells Plate CHO-K1 cells at a density of cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate hours before transfection (65 75% confluency). Incubate cell cultures overnight. 9 Allow X-tremeGENE 9 Transfection Reagent, and Place 200 µl diluent in a sterile tube. Add 6 µl X-tremeGENE 9 Transfection Reagent to the diluent. Add 2 µg plasmid. (3:1 ratio of reagent to ). Add 5 µl transfection complex to the cells in a dropwise Experimental result: CHO-K1 cells were successfully transfected with pc3.1-gfp plasmid using using X-tremeGENE 9 Transfection Reagent.
8 Commonly Used Cell Types 8 COS-7 Cells Plate COS-7 cells at a density of cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate hours before transfection (75 85% confluency). Incubate cell cultures overnight. 9 Allow X-tremeGENE 9 Transfection Reagent, and Place 200 µl diluent in a sterile tube. Add 6 µl X-tremeGENE 9 Transfection Reagent to the diluent. Add 2 µg plasmid. (3:1 ratio of reagent to ). Add 5 µl transfection complex to the cells in a dropwise Experimental result: COS-7 cells were successfully transfected with pc3.1-gfp plasmid using X-tremeGENE 9 Transfection Reagent.
9 Commonly Used Cell Types 9 HeLa Cells Plate HeLa cells at a density of cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate hours before transfection (70 90% confluency). Incubate cell cultures overnight. 9 Allow X-tremeGENE 9 Transfection Reagent, 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 µl diluent in a sterile tube. Add 6 µl X-tremeGENE 9 Transfection Reagent to the diluent. Add 2 µg plasmid. (3:1 ratio of reagent to ). Add 5 µl transfection complex to the cells in a dropwise Experimental result: HeLa cells were successfully transfected with pc3.1-gfp plasmid using X-tremeGENE 9 Transfection Reagent.
10 Commonly Used Cell Types 10 NIH-3T3 Cells Plate NIH-3T3 cells at a density of cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate hours before transfection (70 80% confluency). Incubate cell cultures overnight. 9 Allow X-tremeGENE 9 Transfection Reagent, and Place 200 µl diluent in a sterile tube. Add 6 µl X-tremeGENE 9 Transfection Reagent to the diluent. Add 2 µg plasmid. (3:1 ratio of reagent to ). Add 5 µl transfection complex to the cells in a dropwise Experimental result: NIH-3T3 cells were successfully transfected with pc3.1-gfp plasmid using X-tremeGENE 9 Transfection Reagent.
11 Commonly Used Cell Types 11 HEK-293 Cells Plate HEK-293 cells at a density of cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate hours before transfection (50 70% confluency). Incubate cell cultures overnight. Allow X-tremeGENE HP Transfection Reagent, and Place 200 µl diluent in a sterile tube. Add 2 µg plasmid. HP Add 6 µl X-tremeGENE HP Transfection Reagent to the diluted. (3:1 ratio of reagent to ). Add 10 µl transfection complex to the cells in a dropwise Experimental result: HEK-293 cells were successfully transfected with pc3.1-gfp plasmid using X-tremeGENE HP Transfection Reagent.
12 Difficult-to-Transfect Cells 12 PC-3 Cells Plate PC-3 cells at a density of cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate hours before transfection (75 85% confluency). Incubate cell cultures overnight. Allow X-tremeGENE HP Transfection Reagent, and Place 200 µl diluent in a sterile tube. Add 2 µg plasmid. HP Add 2 µl X-tremeGENE HP Transfection Reagent to the diluted. (1:1 ratio of reagent to ). Add 10 µl transfection complex to the cells in a dropwise Experimental result: PC-3 cells were successfully transfected with pc3.1-gfp plasmid using X-tremeGENE HP Transfection Reagent.
13 Difficult-to-Transfect Cells 13 HCT 116 Cells Plate HCT 116 cells at a density of cells/well. Plate cells in a volume of 1 ml complete growth medium per well in a 12-well plate hours before transfection (80% confluency). Incubate cell cultures overnight. Allow X-tremeGENE HP Transfection Reagent, and Place 100 µl diluent in a sterile tube. Add 1 µg plasmid. HP Add 2 µl X-tremeGENE HP Transfection Reagent to the diluted. (2:1 ratio of reagent to ). Add transfection complex to the cells in a dropwise
14 Difficult-to-Transfect Cells 14 A549 Cells Plate A549 cells at a density of cells/well. Plate cells in a volume of 150 μl complete growth medium per well in a 96-well plate hours before transfection (70% confluency). Incubate cell cultures overnight. Allow X-tremeGENE HP Transfection Reagent, and Place 500 µl diluent in a sterile tube. Add 5 µg plasmid. HP Add 10 µl X-tremeGENE HP Transfection Reagent to the diluted. (2:1 ratio of reagent to ). Add 15 µl transfection complex to the cells in a dropwise
15 Difficult-to-Transfect Cells 15 MCF-7 Cells Plate MCF-7 cells at a density of cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate hours before transfection (70 90% confluency). Incubate cell cultures overnight. 9 Allow X-tremeGENE 9 Transfection Reagent, and Place 200 µl diluent in a sterile tube. Add 6 µl X-tremeGENE 9 Transfection Reagent to the diluent. Add 2 µg plasmid. (3:1 ratio of reagent to ). Add 10 µl transfection complex to the cells in a dropwise Experimental result: MCF-7 cells were successfully transfected with pc3.1-gfp plasmid using X-tremeGENE 9 Transfection Reagent.
16 Difficult-to-Transfect Cells 16 HuH-7 Cells Plate HuH-7 cells at a density of cells/well. Plate cells in a volume of 2.5 ml complete growth medium per well in a 6-well plate hours before transfection. Incubate cell cultures overnight. Allow X-tremeGENE HP Transfection Reagent, and Place 100 µl diluent in a sterile tube. Add 1.5 µg plasmid. HP Add 1.5 µl X-tremeGENE HP Transfection Reagent to the diluted. (1:1 ratio of reagent to ). One hour before adding the complexes to cells, refresh the medium with 1.5 ml of Opti-MEM I Reduced Serum Medium. Add 100 µl transfection complex to the cells in a dropwise The next day refresh the medium with complete growth medium.
17 Difficult-to-Transfect Cells 17 U-2 OS Cells Plate U-2 OS cells at a density of cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate hours before transfection (70 90% confluency). Incubate cell cultures overnight. Allow X-tremeGENE HP Transfection Reagent, and Place 200 µl diluent in a sterile tube. Add 2 µg plasmid. HP Add 4 µl X-tremeGENE HP Transfection Reagent to the diluted. (2:1 ratio of reagent to ). Add 10 µl transfection complex to the cells in a dropwise Experimental result: U-2 OS cells were successfully transfected with pc3.1-gfp plasmid using X-tremeGENE HP Transfection Reagent.
18 Difficult-to-Transfect Cells 18 HepG2 Cells Plate HepG2 cells at a density of cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate hours before transfection (60 70% confluency). Incubate cell cultures overnight. 9 Allow X-tremeGENE 9 Transfection Reagent, and Place 500 µl diluent in a sterile tube. Add 30 µl X-tremeGENE 9 Transfection Reagent to the diluent. Add 10 µg plasmid. (3:1 ratio of reagent to ). Add 5 µl transfection complex to the cells in a dropwise Experimental result: HepG2 cells were successfully transfected with pc3.1-gfp plasmid using X-tremeGENE 9 Transfection Reagent.
19 Difficult-to-Transfect Cells 19 Neuro-2a Cells Protocol provided by a customer. Plate Neuro-2a cells at a density of cells/well. Plate cells in a volume of 500 μl complete growth medium per well in a 24-well plate 24 hours before transfection (30 40% confluency). Incubate cell cultures overnight. 9 Allow X-tremeGENE 9 Transfection Reagent, and Place 100 µl diluent in a sterile tube. Add 8 µl X-tremeGENE 9 Transfection Reagent to the diluent. Add 2 µg plasmid. (4:1 ratio of reagent to ). Add 100 µl transfection complex to the cells in a dropwise 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 defining the criteria for the performance of the specific 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. Experimental result: Neuro-2a cells were successfully transfected with CMV6-GFP plasmid using X-tremeGENE 9 Transfection Reagent.
20 Primary Cells and Stem Cells 20 Primary MEF Cells Plate primary MEF cells at a density of cells/well. Plate cells in a volume of 1 ml complete growth medium per well in a 12-well plate hours before transfection (60% confluency). Incubate cell cultures overnight. Allow X-tremeGENE HP Transfection Reagent, and Place 100 µl diluent in a sterile tube. Add 1 µg plasmid. HP Add 4 µl X-tremeGENE HP Transfection Reagent to the diluted. (4:1 ratio of reagent to ). Add transfection complex to the cells in a dropwise
21 Primary Cells and Stem Cells 21 Human Primary Fibroblasts from Pre-Skin Plate human primary fibroblast cells at a density of cells/well. Plate cells in a volume of 2 ml complete growth medium per well in a 6-well plate hours before transfection (40 50% confluency). Incubate cell cultures overnight. Allow X-tremeGENE HP Transfection Reagent, and Place 200 µl diluent in a sterile tube. Add 2 µg plasmid. HP Add 6 µl X-tremeGENE HP Transfection Reagent to the diluted. (3:1 ratio of reagent to ). Add 200 µl transfection complex to the cells in a dropwise Experimental result: Human primary fibroblasts were successfully transfected with GFP-encoding plasmid using X-tremeGENE HP Transfection Reagent.
22 Primary Cells and Stem Cells 22 Murine Mesenchymal Stem Cells EK8 Plate EK8 cells at a density of cells/well. Plate cells ( cells/ml) in a volume of 100 μl complete growth medium per well in a 96-well plate 24 hours before transfection (50% confluency). Incubate cell cultures overnight. Allow X-tremeGENE HP Transfection Reagent, and Place 392 µl diluent in a sterile tube. Add 8 µg plasmid. HP Add 8 µl X-tremeGENE HP Transfection Reagent to the diluted. (1:1 ratio of reagent to ). Add 10 µl transfection complex to the cells in a dropwise
23 Primary Cells and Stem Cells 23 Human Mesenchymal Stem Cells Plate hmscs at a density of cells/well. Plate cells in a volume of 2 ml complete growth medium per well in a 6-well plate hours before transfection (50% confluency). Incubate cell cultures overnight. Allow X-tremeGENE HP Transfection Reagent, and Place 200 µl diluent in a sterile tube. Add 2 µg plasmid. HP Add 6 µl X-tremeGENE HP Transfection Reagent to the diluted. (3:1 ratio of reagent to ). Add 200 µl transfection complex to the cells in a dropwise Experimental result: human mesenchymal stem cells were successfully transfected with GFP-encoding plasmid using X-tremeGENE HP Transfection Reagent.
24 Packaging Cells and Insect Cells 24 HEK-293T Cells for Lentiviral Production Plate HEK-293T cells at a density of cells/well. Plate cells in a volume of 2 ml complete growth medium per well in a 6-well plate hours before transfection (60 80% confluency). Incubate cell cultures overnight. Allow X-tremeGENE HP Transfection Reagent, and HP Place 180 µl diluent in a sterile tube. Add 2 µg plasmid. (20 μl of a plasmid mixture in a molar ratio of 1: 2: 2 = library plasmid : packaging plasmid : envelope plasmid). Add 6 µl X-tremeGENE HP Transfection Reagent to the diluted. (3:1 ratio of reagent to ). Add 200 µl transfection complex to the cells in a dropwise Experimental result: HEK-293T cells were successfully transfected with pgipz-egfp plasmid using X-tremeGENE HP Transfection Reagent.
25 Packaging Cells and Insect Cells 25 SF9 Insect Cells Plate SF9 cells at a density of 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 Transfection Reagent, and Place 100 µl diluent in a sterile tube. Add 1 µg plasmid. HP Add 8 µl X-tremeGENE HP Transfection Reagent to the diluted. (8:1 ratio of reagent to ). Add transfection complex to the cells in a dropwise For life science research only. Not for use in diagnostic procedures.
26 General QuickProtocols 26 X-tremeGENE 9 Transfection Reagent Cell preparation for transfection Plate cells approx. 24 hours before transfection making sure cells are at optimal concentration (70 90 % confluency). 9 Steps prior to transfection Allow X-tremeGENE 9 Transfection Reagent, and diluent (Opti:MEM I Reduced Serum Medium or serum-free Place diluent in a sterile tube. Add X-tremeGENE 9 Transfection Reagent to the diluent. Add plasmid. Incubate for 15 min at +15 C to +25 C. Add transfection complex to the cells in a dropwise Incubate cells for hours before measuring protein Volumes of X-tremeGENE 9 Transfection Reagent and amounts of for various ratios Ratio Transfection Reagent : Minimal transfection complex volume Volume for a whole 96-well plate Serum-free medium to a final volume of 100 µl 500 µl 3 : 1 X-tremeGENE 9 Transfection Reagent 3 µl 15 µl 1 µg 5 µg 6 : 1 X-tremeGENE 9 Transfection Reagent 6 µl 30 µl 1 µg 5 µg 6 : 2 X-tremeGENE 9 Transfection Reagent 6 µl 30 µl 2 µg 10 µg
27 General QuickProtocols 27 X-tremeGENE HP 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 Transfection Reagent, and diluent (Opti:MEM I Reduced Serum Medium or serum-free Place diluent in a sterile tube. Add plasmid. HP Add X-tremeGENE HP Transfection Reagent to the diluted. Incubate for 15 min at +15 C to +25 C. Add transfection complex to the cells in a dropwise Incubate cells for hours before measuring protein Volumes of X-tremeGENE 9 Transfection Reagent and amounts of for various ratios Ratio Transfection Reagent : Minimal transfection complex volume Volume for a whole 96-well plate Serum-free medium to a final volume of 100 µl 500 µl 1 : 1 X-tremeGENE HP Transfection Reagent 1 µl 5 µl 1 µg 5 µg 2 : 1 X-tremeGENE HP Transfection Reagent 2 µl 10 µl 1 µg 5 µg 3 : 1 X-tremeGENE HP Transfection Reagent 3 µl 15 µl 1 µg 5 µg
28 Published by Roche Diagnostics GmbH Sandhofer Straße Mannheim Germany 2013 Roche Diagnostics. All rights reserved. x-tremegene.roche.com
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