LipoPlas™ Transfection Reagent

CatNo.:IC-6018

Description

LipoPlas is a newly developed and proprietary reagent for the transfection of nucleic acids into eukaryotic cells. LipoPlas has the following advantages:

A.The highest transfection efficiency in many cell types and formats.

B.DNA-LipoPlas complexes can be directly added to cells in culture medium (with or without serum).

C.It is not necessary to remove DNA-LipoPlas complexes or change medium following transfection. The complexes can be removed after 4-6 hours by replacing with fresh medium (optional).

D.Not recommended for RNA transfection.

Storage

Store at 4^oC. DO NOT FREEZE.

Size

0.05 ml;0.75 ml;1.5 ml

Important Guidelines

Follow these guidelines when performing transfections:

1.The ratio of DNA (μg): LipoPlas (μl) to use when preparing complexes should be 1:2 to 1:3 for most cell lines. To transfect 0.3-2 X105 cells in a 24-well format, use 0.5-1 μg DNA and 1-3 μl of LipoPlas. Optimizing transfection by varying DNA/LipoPlas ratio is possible.

2.For most cell lines, 60-80% confluence at the time of transfection is recommended to obtain high efficiency and expression levels and to minimize decreased cell growth associated with high transfection activity. Other cell densities between 50-90% may be used to optimize transfection efficiency and minimize toxicity. Take care to maintain a standard seeding protocol between experiments because transfection efficiency is dependent on culture confluence.

3.It is not necessary to use antibiotic-free medium during transfection. For better results, you may choose to: Use Opti-MEM I medium to dilute LipoPlas prior to complexing with DNA. Other media without serum (e.g. DMEM) may be used to dilute LipoPlas, but transfection efficiency may be compromised.

Note: Some serum-free formulations may inhibit LipoPlas mediated transfection, for example: CD 293, 293 SFM II and VP-SFM etc.

Transfection Procedure for 24-Well Format:

For adherent cells: One day before transfection, plate cells in growth medium so that they will be 60-80% confluence at the time of transfection (0.3-2 x 105 cells/well for a 24-well plate).

For suspension cells: On the day of transfection just prior to preparing complexes, plate 4-8 x 105 cells/ 500 μl of growth medium in a 24-well plate.

1.For each transfection sample, prepare DNA-LipoPlas complexes as follows:

A.Dilute DNA in 25 μl of Opti-MEM I medium without serum (or other medium without serum). Mix gently.

B.Mix LipoPlas gently before use，then dilute the appropriate amount in 25 μl of Opti-MEM I Medium (or other medium without serum). Mix gently and incubate for 5 minutes at room temperature.

Note: Combine the diluted LipoPlas with the diluted DNA within 30 minutes. Longer incubation time may decrease activity. If DMEM is used as a diluent for the LipoPlas, mix with the diluted DNA within 5 minutes.

C. After the 5 minute incubation, combine the diluted DNA with the diluted LipoPlas (total volume  is 50 μl). Mix gently and incubate for 20 minutes at room temperature to allow the DNA-LipoPlas complexes to form. The solution may appear cloudy，but this will not inhibit the transfection.

Note: DNA-LipoPlas complexes are stable for at least 5 hours at room temperature.

2.Add the 50 μl of DNA-LipoPlas complexes to each well. Mix gently by rocking the plate back and forth.

3.Incubate the cells at 37oC in a CO2 incubator for 24-48 hours until they are ready to assay for transgene expression. It is not necessary to remove the complexes or change the medium; however, growth medium may be replaced after 4-6 hours without loss of transfection activity.

For stable cell lines: Passage the cells at a 1:10 or higher dilution into fresh growth medium 24 hours after transfection. Add selective drugs into culture medium after cells attachment to the plate.

Scaling Up or Down Transfections

To transfect cells in different tissue culture formats, vary the amounts of LipoPlas, DNA, cells, and medium used in proportion to the different surface area (see table below). With automated, high-throughput systems, larger  complexing volumes are recommended for transfections in 96-well plates. Note: You may perform rapid 96-well plate transfections (plate cells and transfect simultaneously) by adding a suspension of cells directly to complexes prepared in the plate. Prepare complexes and add cells at twice the cell density as in the basic protocol in a 100 μl volume. Cells will adhere as usual in the presence of DNA-LipoPlas complexes.

Optimizing Transfection

To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying DNA and LipoPlas concentrations, and cell number. Adopt cell density between 60-80% and vary DNA (μg): LipoPlas (μl) ratios from 1: 0.5 to 1:5.

Product Qualification

LipoPlas has been extensively tested in easy transfection cell lines, such as: HEK293, MCF7 cells, and difficult transfection cells, e.g. mouse trophoblast stem cell (TS) and C2C12 cells with an EGFP reporter plasmid. LipoPlas is free of microbial contamination.

Limited Use Restriction

The purchase of this product conveys to the buyer the non-transferable right to use the product and components of the product in research conducted by the buyer. The buyer cannot sell or otherwise transfer this product or its components to a third party and in particular, no rights are conveyed to the buyer to use the product or its components for commercial purposes. Commercial purposes means any activity for which a party receives consideration and may include, but is not limited to:

A.Using of the product or its components in manufacturing;

B.Using of the product or its components to provide a service, information or data;

C.Using of the product or its components for diagnostic purposes;

D.Reselling the product or its components, whether or not such product or its components are resold for use in research.