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Protocol for in vitro Transfection with TurboFect™ Transfection Reagent in a 24-well Plate

Reagents to be supplied by the user:
Serum free DMEM, RPMI or other growth medium. The presence of antibiotics in the medium has no effect on transfection efficiency.

Quantities and volumes should be scaled up according to the number of cells/wells to be transfected. (See Table 1 for scale-up ratios).

(A) Preparation of cells
Plate ~5x10⁴ adherent cells or ~5x10⁵ suspension cells per well 24 h prior to transfection.

Note

• The recommended confluency for adherent cells on the day of transfection is 50-70%.
• Suspension cells should be in logarithmic growth phase at the time of transfection.

(B) Preparation of the TurboFect™/DNA Complexes
Prepare immediately prior to transfection.

1. Dilute 1 µg of DNA in 100 µl of serum free DMEM or other growth medium.
2. Add 2 µl of TurboFect™ to the diluted DNA and mix the solution by pipetting.
3. Incubate 15-20 minutes at room temperature.

Note

• We recommend starting with 1 µg of DNA and 2 µl of TurboFect™ per well of 24-well plate (see Table 1).
• Subsequent optimization may further increase the transfection efficiency depending on the cell line and transgene.

(C) Transfection

1. Add 100 µl of the TurboFect™/DNA mixture drop-wise to each well.
2. Gently rock the plate to achieve even distribution of the complexes.
3. Incubate at 37°C in a CO₂ incubator.
4. Analyze transgene expression 24-48 hours later.

Note
For stable transfection cells should be grown in selective medium for 10-15 days.

Table 1. Scale-up ratios for transfection with TurboFect™ in vitro Transfection Reagent.

Tissue culture vessel	Growth area, cm2/well	Volume of media, ml	Adherent cells to seed the day before transfection*	Amount of DNA		Volume of TurboFect™, µl**
				µg**	µl***	Recommended	Range
96-well plate	0.3	0.2	0.5-1.2 x 104	0.2	20	0.4	0.3-0.6
48-well plate	0.7	0.5	1-3 x 104	0.5	50	1	0.5-1.4
24-well plate	2	1	2-6 x 104	1	100	2	1-2.8
12-well plate	4	2	0.4-1.2 x 105	2	100	4	2-6
6-well plate	9.5	4	0.8-2.4 x 105	4	400	6	4-8
60 mm plate	20	6	2-6.3 x 105	6	600	12	8-16

* These umbers were determined using HeLa cells. Actual value depends on the cell type.
** Amount of DNA and TurboFect™ in vitro Transfection Reagent used may require optimization.
*** The volume of the DNA solution should represent 1/10 of the total volume of the culture medium.

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Updated sausio 30, 2008 13:37