Description

Lip2000™ is a newly developed ａnd proprietary reagent for the transfection of nucleic acids into

eukaryotic cells.

Lip2000™ has the following advantages:

The highest transfection efficiency in many cell types ａnd formats.

DNA-Lip2000™ complexes can be directly added to cells in culture medium (with or without serum).

It is not necessary to remove DNA-Lip2000™ complexes or change medium following transfection.

The complexes can be removed after 4-6 hours by replacing with refresh medium (optional)

Contents ａnd Storage

Lip2000™ is supplied in liquid form at a concentration of 1mg/ml. Store at 4℃. DO NOT FREEZE.

Product Qualification

Lip2000™ has been extensively tested by transfection of HEK293 cells with an EGFP reporter containing

plasmid. Lip2000™ is free of microbial contamination.

Important Guidelines

Follow these guidelines when performing transfections:

1. The ratio of DNA (in μg) : Lip2000™ (in μl) to use when preparing complexes should be 1:2 to 1:3 for

most cell lines. To transfect 0.5 -2 ×105 cells in a 24-well format, use 0.8-1 μg DNA ａnd 2-3 μl of

Lip2000™. Optimizing transfection by varying DNA/Lip2000™ ratio is possible.

2. It is CRITICAL to transfect cells at high cell density. 90-95% confluence the time of transfection is

recommended to obtain high efficiency ａnd expression levels ａnd to minimize decreased cell growth

associated with high transfection activity. Lower cell densities are suitable with optimization of conditions.

Take care to maintain a standard seeding protocol between experiments because transfection efficiency is

dependent on culture confluence.

3. DO NOT add antibiotics to media during transfection as this will cause cell death.

For better results, you may choose to:

Use Opti-MEM I medium to dilute Lip2000™ prior to complexing with DNA. Other media without serum

(e.g.DMEM) may be used to dilute Lip2000™,but transfection efficiency may be compromised.

Note: Some serum-free formulations can inhibit Lip2000™ 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 (without antibiotics) so that

they will be 90-95% confluent at the time of transfection (0.5 -2 ×105 cells/well for a 24-well plate).

For suspension cells: On the day of transfection just prior to preparing complexes,plate 4-8×105cells/500

μl of growth medium (without antibiotics) in a 24-well plate.

1. For each transfection sample, prepare DNA-Lip2000™ complexes as follows:

• Dilute DNA in 50 μl of Opti-MEM I Reduced Serum Medium without serum (or other medium without

serum). Mix gently.

• Mix Lip2000™ gently before use, then dilute the appropriate amount in 50 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 Lip2000™ with the diluted DNA within 30 minutes. Longer incubation times

may decrease activity. If DMEM is used as a diluent for the Lip2000™, mix with the diluted DNA within

5 minutes. After the 5 minute incubation,combine the diluted DNA with the diluted Lip2000™ (total

volume is 100 μl).

•Mix gently ａnd incubate for 20 minutes at room temperature to allow the DNALip2000™ complexes to

form. The solution may appear cloudy,but this will not inhibit the transfection.

Note:DNA-Lip2000™ complexes are stable for at least 5 hours at room temperature.

2. Add the 100 μl of DNA-Lip2000™ complexes to each well. Mix gently by rocking the plate back and

forth.

3. Incubate the cells at 37℃ 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 medium the following day.

For suspension cells: Add PMA and/or PHA (if desired) 4 hours after adding the DNA-Lip2000™

complexes to the cells.

Tip: For Jurkat cells, adding PHA-L ａnd PMA at final concentrations of 1 μg/ml ａnd 50 ng/ml,

respectively, enhances CMV promoter activity ａnd gene expression. For K562 cells, adding PMA alone is

sufficient to enhance promoter activity.

Scaling Up or Down Transfections

To transfect cells in different tissue culture formats, vary the amounts of Lip2000™ , DNA, cells, and

medium used in proportion to the difference in surface area (see table below). With automated,

highthroughput 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.