Transfection protocol

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Eukaryotic transfection protocol:

Principles:

The prevelant transfection method refers to a liposome (plasmids artificially involved) transportation pathway. Liposome is a artificial vesicle packaged by lipid bilayer. Most transfection kits are manufactured on base of this 'lipofection'. Liposome can fuse wtih the cell membrane very well and has high efficiency of transfection.

Materials:

Transfection reagent (Sinofection), pre-cultured cell lines, sterilized pipet and tips

Procedure:

1. Dilute the required plasmid DNA amount with appropriate diluent, such as PBS with Mg2+, 150mM NaCl, 300mM glucose, DMEM medium with or without serum, whichever is isotonic. Incubate for 5 min at room temperature. Add the required Sinofection volume (refered as below) to the diluted DNA and mix gently to assure a total volume of 1.5~3ml.

Culture vessel

Vol. of medium(ml)

DNA(μg/well)

Vol. of Sinofection(μl/well)

96-well plate

0.1

0.05~0.2

0.25~0.75

48-well plate

0.2

0.1~0.4

0.5~1.5

24-well plate

0.5

0.2~0.8

1~3

12-well plate

1

0.4~1.6

2~6

6-well plate

2

0.8~3.2

4~12

 

2. Incubate the mixture for 10-20 minutes at room temperature to allow complexes to form. Do not incubate for longer than 20 minutes.

3. Slowly add 1.5~3ml of DNA-lipid mixture into the wells while slowly swirling the plate.

4. Incubate transfected cell cultures at 37°C for 6 hours and renew the medium

5. Renew the medium for every other day.