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.
规格:0.75ml, 1.5ml, 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. Prepare complexes and add
cells at twice the cell density as
注意：
1. 本产品仅供科研使用。请勿用于医药、临床诊断或治疗，食品及化妆品等用途。请勿存放于普通住宅区。
2. 为了您的安全和健康，请穿好实验服并佩戴一次性手套和口罩操作。