Lipo3K Transfection Reagent

Description

Lipo3K  is a newly developed and proprietary reagent for the transfection of nucleic acids into eukaryotic cells. Lipo3K has the following advantages: 

The highest transfection efficiency in many cell types and formats. 

DNA-Lipo3K complexes can be directly added to cells in culture medium (with or without serum). 

It is not necessary to remove DNA-Lipo3K complexes or change medium following transfection. 

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

Contents and Storage 

     Lipo3Kis supplied in liquid form at a concentration of 1mg/ml.   Store at 4^oC.DO NOT FREEZE.

Product Qualification

     Lipo3K has been extensively tested by transfection of HEK293 cells with an EGFP reporter containing  plasmid. Lipo3K is free of microbial contamination.

Important Guidelines

Follow these guidelines when performing transfections:

1.     The ratio of DNA(inµg) :Lipo3K (in µl) to use when preparing complexes should be 1:2 to1:3 for most celllines. To transfect 0.5 -2 X10⁵cells in a 24-well format, use 0.8-1 µgDNA and 2-3 µl of Lipo3K. Optimizing transfection by varying DNA/Lipo3K ratio is possible. 

2.   It is CRITICALto transfect cells at high celld ensity.90-95% confluence the time of transfection is recommended to obtain high efficiency and expression levels and tominimize 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-MEMI medium to dilute Lipo3K prior to complexing with DNA. Other media without

serum (e.g.DMEM)may be used to dilute Lipo3K，but transfection efficiency may be compromised.

         Note:  Some serum-free formulations can inhibit Lipo3K mediated transfection, for example: CD293，293SFMII，andVP-SFMetc.

Transfection  Procedure for 24-Well Format

 For adherent cells: One day before transfection, plate cells in growth medium(without antibiotics) so thatt hey will be 90-95% confluent at the time o ftransfection(0.5-2x105 cells/well for a 24-well plate).

 Fors uspension cells:On the day of transfection just prior to preparing complexes, plate 4-8x105 cells/500µL of growth medium (without antibiotics) in a 24-well plate.

  1.     For each transfection sample，prepare DNA-Lipo3K complexes as follows:  Dilute DNA in 50µL of Opti-MEMI Reduced Serum Medium without serum (or other medium without serum).Mix gently.

2    MixLipo3K gentlybeforeuse，then dilute the appropriate amount in 50µL of Opti-MEM I Medium (or other medium without serum). Mix gently and incubate for 5minutes at room temperature.

Note:Combine the diluted Lipo3K with the diluted DNA within 30 minutes. Longer incubation times may decrease activity. If DMEM is used as adiluent for the Lipo3K ，mix with the diluted DNA with in 5minutes.

After the 5minute incubation, combine the diluted DNA with the diluted Lipo3K (total volumeis100µL). Mix gently and incubate for 20 minutes at roomtemperature to allow theDNA-Lipo3K  complexes to form.The solution may appear cloudy, but this will not inhibit the transfection.Note:DNA-Lipo3K complexes are stable for at least 5hours at roomtemperature. 

3.      Add the100µl of DNA-Lipo3Kcomplexes to each well.Mix gently by rocking the plate back and forth.

4.      Incubate the cells at 37^oC in a CO2 incubator for 24-48hours 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 4-6hours without loss of transfection activity.

For stable celllines:Passage the cells at a1:10 or higher dilution int ofresh growth medium 24hours after transfection. Add selective medium the following day.

For suspension cells:Add PMA and/ or PHA (if desired) 4hours after adding the DNA-Lipo3K complexes to the cells. Tip:For Jurkat cells，adding PHA-L and PMA at final concentrations of 1µg/ml and 50ng/ml，respectively，enhances CMV promoter activity and gene expression. ForK 562 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 Lipo3K，DNA，cells，and medium used in proportion to the difference in 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 rapid96-well plate transfections (plate cells and transfects imultaneously) 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 100µl volume.Cells will adhere as usual in the presence of DNA-Lipo3K complexes.

Note:Surface areas are determined from actual measurements of tissue culture vessels.

Optimizing  Transfection

    To obtain the highest transfection efficiency and low non-specificeffects, optimize transfection conditions by varying DNA and Lipo3K concentrations，and cellnumber. Make sure that cells are greater than 90% confluent and varyDNA (µg) : Lipo3K (µl) ratios from1:0.5 to1:5.

NOTE: Always wear lab coats, gloves and goggles when working with our products although they are low-risk chemicals for R&D only.