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Transfection

TurboFect™ Transfection Reagent

Product information

Features

• High transfection efficiency of a wide variety of cell types:
  – primary and secondary cell lines,
  – adherent and suspension cells,
  – differentiated and undifferentiated cells,
  – stable and transient transfections.
• No optimization required for most cell lines.
• Excellent transfection efficiency in the presence or absence of serum.
• Minimal cytotoxicity.
• Ready-to-use reagent – no need to reconstitute, dilute or manipulate.
• Simple protocol.

Applications

• Plasmid DNA and oligonucleotide transfection.
• mRNA transfection.
• Co-transfection.
• Transfection of primary and secondary cell lines.
• Stable and transient transfection.

Description

TurboFect™ in vitro Transfection Reagent is a sterile solution of a proprietary cationic polymer in water. The polymer forms compact, stable, positively charged complexes with DNA. These complexes protect DNA from degradation and facilitate gene delivery into eukaryotic cells (see Fig.1). TurboFect™ is ideal for transfection of a variety of cells, including primary and difficult-to-transfect cells (see Fig.2 and list of cells below). Transfection can be performed in the presence or absence of serum. TurboFect™ demonstrates superior transfection efficiency and minimal toxicity when compared to lipid-based or other polymer-based transfection reagents (see Fig.1).

Storage

Store at 4°C.

Successfully Transfected Cells

Permanently growing cell lines

B50 rat nervous tissue neuronal cells
BAEC bovina aorta endothelial cells (customer feedback)
BHK 21 baby hamster kidney cells
Calu1 human lung epidermoid carcinoma cells
CHO chinese hamster ovary cells
COLO human colon adenocarcinoma cells
Cos-7 african green monkey kidney cells
HCT116 human colorectal cancer cells (customer feedback)
HEK293 human embryonic kidney cells
HEL299 cultured human embryonic lung cells (customer feedback)
HeLa human cervix adenocarcinoma cells
HeLa S3 human cervix carcinoma cells
Hep2C human larynx carcinoma cells
HepG2 human hepatoma cells
Jurkat human leukaemic T cells
L929 mouse connective tissue fibroblasts
MDCK Madin Darby Canine Kidney cells
MEF mouse embryonic fibroblast cells (customer feedback)
MPC5 conditionally immortalized mouse podocyte cell line (customer feedback)
NIH3T3 mouse embryo fibroblasts
NTEPA2 human neuronal precursor cell line (customer feedback)
RAW264 mouse leukaemic monocyte-macro phage cells
RBL-2H3 rat basophilic leukemia cells (customer feedback)
Sp2/Ag14 mouse myeloma cells
STHdh^Q7/Q7 mouse striatal cell lines
STHdh^Q111/Q111 mouse striatal cell lines
WEHI mouse B cell lymphoma cells

Primary cell cultures

HLF human lung fibroblasts
Mouse bone marrow derived dendritic cells
Mouse bone marrow derived macrophages
Mouse lymphoma cell primary culture (customer feedback)
Primary human muscle cells (customer feedback)
Rat embryonic fibroblasts

Figure 1. EGFP expression in cell lines transfected with TurboFect™ in vitro Transfection Reagent.
A – primary mouse dendritic cells derived from bone marrow,
B – HeLa cells

Figure 2. Comparison of Fermentas TurboFect™ in vitro transfection reagent with tranfection reagents from other vendors.
HeLa cells were transfected with plasmid DNA encoding eGFP. Transfections were performed according to manufacturers recommendations and analysed by flow cytometry.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

General Considerations for Transfection

I. DNA Quality Requirements.
DNA quality is critical for successful transfection. Endotoxin-contaminated DNA may result in inefficient transfection and cause unacceptably high cellular toxicity. For DNA an A₂₆₀/A₂₈₀ ratio of 1.8, or greater is recommended.
II. Cell Density.
The recommended confluency for adherent cells on the day of transfection is 50-70% and 70-90% for TurboFect™ reagents. Suspension cells should be plated at an optimal density ensuring their logarithmic growth at the time of transfection.
III. Incubation Time.
Transient transgene expression takes place within 2-72 hours after DNA transfection. The optimal time depends on the cell type, promoter strength and expression product, and has to be determined experimentally. The recommended incubation time of cells with TurboFect™/protein complexes is 2 hours.
IV. Choice of Promoter.
High transfection efficiency depends both on the transgene promoter and on the cell line used. The cytomegalovirus (CMV) promoter is commonly used for high gene expression in a variety of cell lines. Other promoters, such as those from simian virus (SV40) and from Rous sarcoma virus (RSV) can also be used.
V. Transfection Reagent/Biomolecule Ratio.
The amount of transfection reagent used in transfection depends on the amount of DNA, siRNA or protein and cells to be transfected. The ratios presented in the protocols are starting ratios and can be further optimized for the best results.
VI. Transfection in the Presence of Serum.
Nucleic acid transfection efficiency using Fermentas transfection reagents is consistently high in the presence of serum. The presence of serum may reduce protein transfection efficiency by up to 50%. Therefore, protein transfection in serum-free medium is recommended for best results.
VII. Centrifugation.
Gentle centrifugation of tissue culture plates for 5 minutes at 280 x g after addition of the polyplexes can improve transfection efficiency.

In vitro DNA Transfection using TurboFect™ in vitro Transfection Reagent

Reagents to be Supplied by the User: serum free DMEM, RPMI or other growth medium. The presence of antibiotics in the medium has no effect on transfection efficiency.
The protocol below is provided for 24-well plate.
Quantities and volumes should be scaled up according to the number of cells/wells to be transfected (see Table below for scale-up ratios).

1. In each well, seed ~5x10⁴ adherent cells or ~5x10⁵ suspension cells 24 h prior to transfection.
   

   Note

   • The recommended confluency for adherent cells on the day of transfection is 50-70%.
   • Suspension cells should be in logarithmic growth phase at the time of transfection.
2. Dilute 1 µg of DNA in 100 µl of serum free DMEM or other growth medium.
3. Add 2 µl of TurboFect™ to the diluted DNA and mix the solution by pipetting.
4. Incubate 15-20 minutes at room temperature.
   

   Note

   • Prepare immediately prior to transfection.
   • We recommend starting with 1 µg of DNA and 2 µl of TurboFect™ per well in a 24-well plate (see Table below).
   • Subsequent optimization may further increase transfection efficiency depending on the cell line and transgene used.
5. Add 100 µl of the TurboFect™/DNA mixture drop-wise to each well. Do not remove the growth medium from the cells.
6. Gently rock the plate to achieve even distribution of the complexes.
7. Incubate at 37°C in a CO₂ incubator.
8. Analyze transgene expression 24-48 hours later.
   For stable transfection cells should be grown in selective medium for 10-15 days.
   Plates can be centrifuged for 2-5 min at 200xg to facilitate sedimentation of cells to the bottom of the plate.

Table. Scale-up ratios for transfection with TurboFect™ in vitro Transfection Reagent.

Tissue culture vessel	Growth area, cm2/well	Media, ml	Adherent cells to seed the day before transfection*	DNA, µg**(µl***)	Volume of TurboFect™ (µl)**
					Recommended	Range
96-well plate	0.3	0.2	0.5-1.20 x 104	0.2 (20)	0.4	0.3-0.6
48-well plate	0.7	0.5	1.0-3.0 x 104	0.5 (50)	1.0	0.5-1.4
24-well plate	2.0	1.0	2.0-6.0 x 104	1.0 (100)	2.0	1.0-2.8
12-well plate	4.0	2.0	0.4-1.2 x 105	2.0 (200)	4.0	2.0-6.0
6-well plate	9.5	4.0	0.8-2.4 x 105	4.0 (400)	6.0	4.0-8.0
60 mm plate	20.0	6.0	2.0-6.3 x 105	6.0 (600)	12.0	8.0-16.0

Note

* These numbers were determined using HeLa cells. Actual value depends on the cell type.
** Amount of DNA and TurboFect™ in vitro Transfection Reagent used may require optimization.
*** The volume of the DNA solution should represent 1/10 of the total volume of the culture medium.