i Trans® Transfection Reagent

1.       What this Product Does

Number of Tests

Using the standard procedure, 1 ml of i Trans® Transfection Reagent can be used to perform up to 1000 transfections in 96-well plates using 4:1

Storage and Stability

Store i Trans® Transfection Reagent at +2 to +8 ℃, with the lid tightly closed. The reagent is stable until the expiration date printed on the label when stored under these conditions.

Additional Equipment and Reagents Required

Additional reagents and equipment required to perform transfection assays using i Trans® Transfection Reagent include:

u  Standard Laboratory Equipment

i.              Standard cell culture equipment (e.g., biohazard hoods, incubators)

ii.              Standard pipettes and micropipettes

u  For Plasmid  or siRNA Preparation

i.              For plasmid: Purified plasmid stock in sterile TE (10 mM Tris, 1 mM EDTA, pH8.0) buffer or sterile water

ii.              For siRNA: siRNA stock in sterile RNase free water

u  For Transfection-Complex Formation

i.              opti-MEM I Reduced Serum Medium or serum-free medium

ii.              Sterile polypropylene tubes or round-bottom 96-well plates

u  Growing cells

i.              Select subconfluent cultures in log phase for preparation of cell cultures

ii.              Quantify cell number to reproducibly plate the same number of cells

Applicaiton

i Trans® Transfection Reagent is a non-liposomal multi-component reagent for experiments in cellular analysis. Its low cytotoxicity ,minimal need for optimization , and ability to provide high transfection efficiency in a wide range of commonly used cell lines even in the presence of serum, are especially suited for applications in cellular and molecular research.

2.       Transient plasmid DNA transfection protocol

Use the following procedure to tranfect mamalian cells in a 24-well format. For other formats, see Scaling Up or Down Transfections. All amounts and volumes are given on a per well basis.

(1)  One day before transfection, plate 0.5-2 X 10⁵ cells in gowth medium without antibiotics so that cells will be 70-90% confluent at the time of transfection.

(2)  Dilute 0.8 ug  of DNA in 50 μl of Opti-MEM Reduced Serum Medium without serum. Mix gently.

(3)  Add 3.2 μl i Trans® Transfection Reagent (4:1 reagent to DNA ratio) and pipet gently to mix completely.

(4)  Incubate for 10-12 minutes at room temperature.

(5)  Add the 50 μl of complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.

(6)  Optimizing Transfection: Change the medium after 8 hours.

Scaling Up or Down Transfections

To transfect cells in different tissue culture formats, vary the amounts of i Trans® Transfection, DNA, cells, and medium used in proportion to the relative amounts and volumes, as shown in the table.