Lipofectamine PLUS Reagent Transfection
Protocol
NOTE: This protocol is designed for
transfection of adherent mammalian cells (ex: ES cells) in one well of a
6-well plate. Adjust the amount of starting materials accordingly if using
more than one well. If you are using a different sized dish, refer to Table 1
for the corresponding starting amounts suggested for the dish you are using.
1.
The
day before transfection, trypsinize and count cells, plating them so that they
are 70% to 90% confluent on the day of transfection. During plating and
transfection, do not use antibiotics (example: PS)- this helps cell growth and
allows transfection without rinsing the cells.
On the actual day of transfection:
2.
In a
1.5 mL microcentrifuge tube, combine 1 mg of DNA (x mL), Complexation
Medium (100-x-6 mL) and 6 mL of Plus Reagent. Mix and incubate at room temperature for
15 min.
3.
In a
second 1.5 mL microcentrifuge tube, combine 4 mL of Lipofectamine Reagent and 96 mL of Complexation Medium.
4.
Combine
pre-complexed DNA from Step 2 with the diluted Lipofectamine Reagent from Step
3. Mix and incubate at room temperature for 15 min.
5.
While
complexes are forming, replace medium on the cells with 800 mL of Transfection Medium per
well.
6.
Add
200 mL of
DNA-PLUS-Lipofectamine Reagent complex for each well. Mix complexes into the
medium gently. Incubate at 37oC with 5% CO2 for 3 hours.
7.
After
3 hours of incubation, add 0.7 to 1.5 mL of Complete Medium (regular culture
medium, contains PS and 20% FCS) to each well.
8.
Depending
on promoter activity and other factors, assay cell extracts for reporter gene
activity 24-72 hours after transfection. For stable expression, passage cells
into fresh culture medium 1 day after transfection and on the next day, add
selection antibiotic. Several days or weeks are required for stable
expression.
Table 1. Suggested starting amounts
of Lipofectamine Plus Reagent transfections for adherent cells
|
|
DNA (mg)
|
PLUS Reagent (mL)
|
Complexation Medium (mL)
|
Lipofectamine Reagent (mL)
|
Transfection Medium (mL)
|
Total Volume (mL)
|
|
Protocol Step
|
2
|
2
|
2 and 3
|
3
|
5
|
6
|
|
96-well plate
|
0.1
|
1
|
10
|
0.5
|
0.05
|
0.07
|
|
24-well plate
|
0.4
|
4
|
25
|
1
|
0.2
|
0.250
|
|
12-well plate
|
0.7
|
5
|
62
|
2.5
|
0.5
|
0.625
|
|
6-well plate
|
1
|
6
|
100
|
4
|
0.8
|
1.0
|
|
60-mm dish
|
2
|
8
|
250
|
12
|
2
|
2.5
|
|
100-mm dish
|
4
|
20
|
750
|
30
|
5
|
6.5
|
Table 2. Complexation
medium
|
|
55 mL recipe
|
|
ES
cell DMEM
|
53.35 mL
|
|
100X
Glutamine
|
550 mL
|
|
100
X Non-essential amino acids
|
550 mL
|
|
LIF
|
5.5 mL
|
|
100X
betamercaptoethanol*
|
550 mL
|
*To make 100X betamercaptoethanol, 7
mL of betamercaptoethanol
to 10 mL of PBS.
Table 3. Transfection medium
|
|
55 mL recipe
|
|
ES
cell DMEM
|
47.85 mL
|
|
FCS
(Final conc‘n: 10%)a
|
5.5 mL
|
|
100X
Glutamine
|
550 mL
|
|
100
X Non-essential amino acids
|
550 mL
|
|
LIF
|
5.5 mL
|
|
100X
betamercaptoethanol*
|
550 mL
|
*To make 100X betamercaptoethanol, 7
mL of betamercaptoethanol
to 10 mL of PBS.
aFor ES cells, performing
lipofection with FCS is recommended for maximum transfection efficiency.
