TransPass™ D1 Transfection Reagent

Application Features

Background:

The introduction of recombinant DNA into cultured cells or "transfection", has become an essential tool for studying gene function and regulation. Initially, cells were transfected by methods such as Calcium Phosphate co-precipitation, electroporation or viral factors (1, 2). The introduction of cationic lipids or polymers as transfection agents has led to the development of reagents that efficiently deliver DNA through the cell membrane and into the nucleus (3).

Transfection Guidelines:

• For consistent results, it is important to maintain healthy proliferating cells that are regularly passaged. 
• It is important that NO heparin and NO antibiotics/antimycotics in the growth medium during transfection.
• Use sterile plasmid DNA that is purified by CsCl gradient centrifugation or column chromatography. 
• The following parameters can be optimized in order to maximize the transfection efficiency for a particular cell line: the cell density at the time of transfection, amount of transfection reagent, amount of plasmid DNA and the culture incubation time before analysis must be optimized.

1. For most cell lines try Protocol 1 first (transfection in the presence of serum). Keep the amount of plasmid DNA constant while varying the amount of TransPass D1 Transfection Reagent. Once the optimal ratio (transfection reagent to DNA) is established, the amount of plasmid DNA and transfection reagent can be increased. Use Table 1 as a guide to optimize the transfection protocol for different plate formats. A convenient method for optimizing transfection conditions is to use pCMV-GLuc 2 Control Plasmid (NEB #N8081), which contains the gene for the secreted Gaussia luciferase.
2. For both Protocols 1 and 2, serum-free medium is required to form the transfection complexes (Step 2).
3. The TransPass D1 Transfection Reagent tube should be vortexed just before use to thoroughly mix the suspension.
4. Multiple plasmids can be combined (step 2) and simultaneously transfected. For consistent results, the total amount of transfected DNA should stay the same between different wells.
5. If you are using Protocol 2 and the cells are sensitive to prolonged absence of serum, the exposure to the serum-free medium can be minimized by decreasing the incubation time (Step 7).
6. For suspension cells, the following protocol (modification of Protocol 1) is recommended: 
   Prepare the plasmid/TransPass D1 complex in 0.5 ml serum-free medium per well of a 12-well plate. Incubate at room temperature for 20-30 minutes. Immediately prior to adding transfection complex (step 5 of Protocol 1), spin down cells to be transfected and resuspend them in high glucose DMEM at a density of 5 x 106 cells per ml. Aliquot 100 µl of cell suspension per well of a 12-well plate and add 0.5 ml of the transfection complex. Rock the plate gently in order to evenly disperse the complex mixture and incubate the cells for two hours. Add complete media with serum as follows: 2 ml per well for a 6-well plate, 1 ml per well for a 12-well plate and 0.5 ml per well for a 24-well plate.

1. Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, (2nd ed.). 1989,
2. Ausubel, F. M. et al. (1987). Current Protocols in Molecular Biology, (2nd ed.).
3. Felgner P.L. et al. (1987). Proc. Natl. Acad. Sci. USA. 84, 7413-7417.

1. TransPass D1 Protocol 1: Transfection in the presence of serum
2. TransPass D1 Protocol 2: Transfection in the absence of serum

1. Cellular Imaging and Analysis FAQs

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