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TransPass™ HeLa Transfection Reagent |
 | | | | TransPass™ HeLa Transfection Reagent | | | |
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Prices are in US dollars and valid only for US orders.
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Description:
The TransPass™ HeLaTransfection Reagent is a lipid/cationic polymer formulation that can be used for efficient transfection of plasmid DNA or siRNA into HeLa cells with a serum-compatible protocol. Co-transfection of plasmid and siRNA can also be efficiently performed.
Figure 1: HeLa cells transfected with a GFP-expressing plasmid using TransPass HeLa Transfection Reagent. The transfection complex mixture is composed of 3 µg plasmid DNA expressing GFP and 9 µl TransPass HeLa Transfection Reagent in 1 ml of serum-free DMEM/high glucose. The transfection complex was added to a well of a 6-well plate containing HeLa cells (approximately 80% confluence), and followed by overnight incubation. Transfected cells were observed microscopically at 20X magnification by either phase (left) or fluorescence (right) microscopy.
Figure 2: Titration of TransPass HeLa Transfection Reagent in HeLa cells using secreted Gaussia Luciferase (pCMV-GLuc Control Plasmid) as a reporter. Cells are transfected in 24-well plate format (approximately 80% confluence) with 0.7 µg pCMV-GLuc in the presence of 10% serum. After 24 hours of transfection, GLuc activity was assayed from 20 µl of the cell supernatant.
Background:
The introduction of recombinant DNA into cultured cells or "transfection", has become an essential tool for studying gene function and regulation. Initially, cells were transfected by methods such as Calcium Phosphate co-precipitation, electroporation or viral factors (1, 2). The introduction of cationic lipids or polymers as transfection agents has led to the development of reagents that efficiently deliver DNA through the cell membrane and into the nucleus (3). However, not all DNA transfection reagents can deliver siRNA as efficiently as plasmid DNA and vice versa.
Transfection Guidelines:
For consistent results, it is important to maintain healthy proliferating cells that are regularly passaged.
Use sterile plasmid DNA that is purified by CsCl gradient centrifugation or column chromatography.
The following parameters can be optimized in order to maximize the transfection efficiency for a particular cell line: the cell density at the time of transfection, amount of transfection reagent, amount of plasmid DNA and the culture incubation time before analysis. The specific amount of transfection reagent can be optimized for a particular cell line by performing a simple titration (Figure 2).
Reaction & Storage Conditions
Storage Temperature:
4°C
Notes
General notes:- TransPass HeLa is a proprietary formulation manufactured by Mirus Bio Corporation and covered by its patents and patents pending.
Usage notes:- In order to form the transfection complexes (protocol step 2), serum-free medium is required.
- For optimal transfection of a certain cell density, the amount of TransPass HeLa Transfection Reagent can be titrated while keeping the amount of plasmid DNA constant. Use Table 1 as a guideline for amounts using different plate sizes. A convenient method for optimizing transfection conditions is to use pCMV-GLuc Control Plasmid (NEB #N8081), which contains the gene for the secreted Gaussia Luciferase. See Figure 2 for an example titration in HeLa cells.
- Plasmids can be combined with siRNA (see protocol step 4) and simultaneously transfected.
- The amount of siRNA to be used needs to be determined. Most siRNAs, such as the Shortcut siRNA Mixes, are effective (maximum silencing) at a final concentration of 2-25 nM. High transfection efficiency is essential for endogenous gene silencing.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Each lot of transfection reagent is tested for efficient delivery of a reporter plasmid and fluorescent siRNA in HeLa cells.
References
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, (2nd ed.), Cold Spring Harbor: Cold Spring Harbor Laboratory.
- Ausubel, F. M. et al. (1987) Current Protocols in Molecular Biology, (2nd ed.), New York: Greene Publishing Associates and Wiley-.
- Felgner P.L. et al. (1987) Proc. Natl. Acad. Sci. USA, 84, 7413-7417.
Companion Products
Gaussia Luciferase Assay Kit
Fluorescein-siRNA Transfection Control
Luciferase Cell Lysis Buffer
pCMV-GLuc Control Plasmid
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