· 别名 : 脂质体2000 转染试剂2000
· 英文名称 : Lip2000™ Transfection
Reagent
· 储存条件 : 4℃,DO NOT FREEZE.
Lip2000™ is a newly developed and proprietary reagent for the
transfection of nucleic acids into eukaryotic cells.
Lip2000™ has the following advantages:
The highest transfection efficiency in
many cell types and formats.
DNA-Lip2000™ complexes can be directly added to
cells in culture medium (with or without serum).
It is not necessary to remove DNA-Lip2000™ complexes or change
medium following transfection.
The complexes can be removed after 4-6
hours by replacing with refresh medium (optional)
Contents and Storage
Lip2000™ is supplied in liquid form at a concentration of
1mg/ml. Store at 4℃. DO NOT FREEZE.
Product Qualification
Lip2000™ has been extensively tested by transfection of HEK293 cells
with an EGFP reporter containing
plasmid. Lip2000™ is free of microbial
contamination.
Important Guidelines
Follow these guidelines when performing
transfections:
1. The ratio of DNA (in μg) : Lip2000™ (in μl) to use when preparing complexes
should be 1:2 to 1:3 for most cell lines. To transfect 0.5 -2 ×105 cells in a 24-well format, use 0.8-1 μg DNA and 2-3 μl of Lip2000™. Optimizing transfection by varying DNA/Lip2000™ ratio is possible.
2. It is CRITICAL to transfect cells at high cell density.
90-95% confluence the time of transfection is recommended to obtain high
efficiency and expression levels and to minimize decreased cell growth
associated with high transfection activity. Lower cell densities are suitable
with optimization of conditions. Take care to maintain a standard seeding
protocol between experiments because transfection efficiency is dependent on
culture confluence.
3. DO NOT add antibiotics to media during
transfection as this will cause cell death.
For better results, you may choose to:
Use Opti-MEM I medium to dilute Lip2000™ prior to complexing
with DNA. Other media without serum (e.g.DMEM) may be used to dilute Lip2000™,but transfection efficiency may be compromised.
Note: Some serum-free formulations can inhibit
Lip2000™ mediated transfection, for example:CD 293, 293 SFM II, and VP-SFM etc.
Transfection Procedure for 24-Well Format
For adherent cells: One day before transfection,plate cells in
growth medium (without antibiotics) so that they will be 90-95% confluent at
the time of transfection (0.5 -2 ×105 cells/well for a 24-well plate).
For suspension cells: On the day of transfection just prior to
preparing complexes,plate 4-8×105cells/500 μl of growth medium
(without antibiotics) in a 24-well plate.
1. For each transfection sample, prepare
DNA-Lip2000™ complexes as follows:
• Dilute DNA in 50 μl of Opti-MEM
I Reduced Serum Medium without serum (or other medium without serum). Mix
gently.
• Mix Lip2000™ gently before use,
then dilute the appropriate amount in 50 μl of Opti-MEM I Medium (or other medium without serum). Mix
gently and incubate for 5 minutes at room temperature.
Note: Combine the diluted Lip2000™ with the diluted DNA
within 30 minutes. Longer incubation times may decrease activity. If DMEM is
used as a diluent for the Lip2000™, mix with the
diluted DNA within 5 minutes. After the 5 minute incubation,combine the diluted
DNA with the diluted Lip2000™ (total volume is 100 μl).
•Mix gently and incubate for 20 minutes at room
temperature to allow the DNALip2000™ complexes to form.
The solution may appear cloudy,but this will not inhibit the transfection.
Note:DNA-Lip2000™ complexes are stable
for at least 5 hours at room temperature.
2. Add the 100 μl of DNA-Lip2000™ complexes to each well. Mix gently by rocking the plate back and
forth.
3. Incubate the cells at 37℃ in a CO2 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.
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-Lip2000™ complexes to the cells.
Tip: For Jurkat cells, adding PHA-L and
PMA at final concentrations of 1 μg/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 Lip2000™ , DNA, cells, and medium used in proportion to the
difference in surface area (see table below). With automated, highthroughput
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.
