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參數(shù)資料
| 型號(hào)： | LIPO |
| 廠商： | Electronic Theatre Controls, Inc. |
| 英文描述： | Lipofectamine 2000 |
| 中文描述： | 脂質(zhì)體2000 |
| 文件頁(yè)數(shù)： | 2/2頁(yè) |
| 文件大?。?/td> | 142K |
| 代理商： | LIPO |
Transfection Procedure
Use the following procedure to transfect mammalian cells in a
24-well format
. To transfect cells in other formats, see
Scaling
Up or Down Transfections
.
Page 2
1.
Adherent cells:
One day before transfection, plate 0.5-2 x 10
5
cells in 500
μ
l of growth medium without antibiotics per
well so that they will be 90-95% confluent at the time of transfection.
Suspension cells:
On the day of transfection just prior to preparing complexes, plate 4-8 x 10
5
cells in 500
μ
l of growth
medium without antibiotics per well.
For each transfection sample
, prepare DNA-Lipofectamine
2000 complexes as follows:
Dilute DNA in 50
μ
l of Opti-MEM
I Reduced Serum Medium without serum (or other medium without serum). Mix
gently.
Mix Lipofectamine
2000 gently before use, then dilute the appropriate amount in 50
μ
l of Opti-MEM
I Medium (or
other medium without serum). Mix gently and incubate for 5 minutes at room temperature.
Note:
Combine the
diluted Lipofectamine
2000 with the diluted DNA within 30 minutes. Longer incubation times may decrease
activity. If D-MEM is used as a diluent for the Lipofectamine
2000, mix with the diluted DNA within 5 minutes.
After the 5 minute incubation, combine the diluted DNA with the diluted Lipofectamine
2000 (total volume is
100
μ
l). Mix gently and incubate for 20 minutes at room temperature to allow the DNA-Lipofectamine
2000
complexes to form. The solution may appear cloudy, but this will not inhibit the transfection.
Note:
DNA-
Lipofectamine
2000 complexes are stable for 6 hours at room temperature.
Add the 100
μ
l of DNA-Lipofectamine
2000 complexes to each well containing cells and medium. Mix gently by rocking
the plate back and forth.
2.
a.
b.
c.
3.
4.
Incubate the cells at 37°C in a CO
2
incubator for 24-48 hours until they are ready to assay for transgene expression. It is
not necessary to remove the complexes or change the medium; however, growth medium may be replaced after 4-6 hours
without loss of transfection activity.
5.
For stable cell lines:
Passage the cells at a 1:10 or higher dilution into fresh growth medium 24 hours after transfection.
Add selective medium the following day.
For suspension cells:
Add PMA and/or PHA (if desired) 4 hours after adding the DNA-Lipofectamine
2000 complexes
to the cells.
Tip:
For Jurkat cells, adding PHA-L and PMA at final concentrations of 1
μ
g/ml and 50 ng/ml, respectively,
enhances CMV promoter activity and gene expression. For K562 cells, adding PMA alone is sufficient to enhance
promoter activity.
Scaling Up or Down Transfections
To transfect cells in different tissue culture formats, vary the amounts of Lipofectamine
2000, DNA, cells, and medium used
in proportion to the difference in surface area (see table). With automated, high-throughput systems, larger complexing
volumes are recommended for transfections in 96-well plates.
Note:
You may perform rapid 96-well plate transfections (plate
cells and transfect simultaneously) by adding a suspension of cells directly to complexes prepared in the plate. Prepare
complexes and add cells at twice the cell density as in the basic protocol in a 100
μ
l volume. Cells will adhere as usual in the
presence of DNA-Lipofectamine
2000 complexes.
Culture
Vessel
per Well (cm
2
)
Area (vs. 24-well)
Medium
Surface Area
Relative Surface
Volume of Plating
DNA (
μ
g) and
Dilution Volume (
μ
l)
0.2
μ
g in 25
μ
l
0.8
μ
g in 50
μ
l
1.6
μ
g in 100
μ
l
4.0
μ
g in 250
μ
l
4.0
μ
g in 250
μ
l
8.0
μ
g in 0.5 ml
24
μ
g in 1.5 ml
Lipofectamine
2000 (
μ
l)
and Dilution Volume (
μ
l)
0.5
μ
l in 25
μ
l
2.0
μ
l in 50
μ
l
4.0
μ
l in 100
μ
l
10
μ
l in 250
μ
l
10
μ
l in 250
μ
l
20
μ
l in 0.5 ml
60
μ
l in 1.5 ml
96-well
0.3
0.2
100
μ
l
500
μ
l
24-well
2
1
12-well
4
2
1 ml
35-mm
10
5
2 ml
6-well
10
5
2 ml
60-mm
20
10
5 ml
10-cm
60
30
15 ml
Note:
Surface areas are determined from actual measurements of tissue culture vessels.
Optimizing Transfection
To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying DNA
and Lipofectamine
2000 concentrations, and cell density. Make sure that cells are greater than 90% confluent and vary
DNA (
μ
g):Lipofectamine
2000 (
μ
l) ratios from 1:0.5 to 1:5.
2000-2002 Invitrogen Corporation. All rights reserved.
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