| 基本信息： | 别名：脂质体2000 转染试剂2000 英文名称：Lip2000 Transfection Reagent 中文名称：脂质体2000/Lip2000 规格：0.75ml ; 1.5ml 储存条件：2-8℃，DO NOT FREEZE |
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Lip2000 is a newly developedand proprietary reagent for the transfection of nucleic acids into eukaryoticcells.
Lip2000 has the followingadvantages:
The highest transfectionefficiency in many cell types and formats.
DNA-Lip2000 complexes can bedirectly added to cells in culture medium (with or without serum).
It is not necessary to removeDNA-Lip2000 complexes or change medium following transfection.
The complexes can be removedafter 4-6 hours by replacing with refresh medium (optional)
Contents andStorage
Lip2000 is supplied in liquidform at a concentration of 1mg/ml. Store at 4℃.DONOT FREEZE.
ProductQualification
Lip2000 has been extensivelytested by transfection of HEK293 cells with an EGFP reporter containing
plasmid. Lip2000 is free ofmicrobial contamination.
ImportantGuidelines
Follow these guidelines whenperforming transfections:
1. The ratio of DNA (in μg) :Lip2000 (in μl) to use when preparing complexes should be 1:2 to 1:3 for mostcell lines. To transfect 0.5 -2 ×105cells in a24-well format, use 0.8-1 μg DNA and 2-3 μl of Lip2000. Optimizingtransfection by varying DNA/Lip2000 ratio is possible.
2. It isCRITICALtotransfect cells at high cell density. 90-95% confluence the time oftransfection is recommended to obtain high efficiency and expression levels andto minimize decreased cell growth associated with high transfection activity.Lower cell densities are suitable with optimization of conditions. Take care tomaintain a standard seeding protocol between experiments because transfectionefficiency is dependent on culture confluence.
3.DO NOTaddantibiotics to media during transfection as this will cause cell death.
For better results, you maychoose to:
Use Opti-MEM I medium to diluteLip2000 prior to complexing with DNA. Other media without serum (e.g.DMEM) maybe used to dilute Lip2000,but transfection efficiency maybe compromised.
Note:Some serum-free formulationscan inhibit Lip2000 mediated transfection, for example:CD 293, 293 SFM II, and VP-SFM etc.
TransfectionProcedure for 24-Well Format
For adherent cells:One day before transfection,plate cells in growth medium (withoutantibiotics) so that they will be 90-95% confluent at the time of transfection(0.5 -2 ×105cells/well for a24-well plate).
For suspension cells: On the day of transfectionjust 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 ofOpti-MEM I Reduced Serum Medium without serum (or other medium without serum).Mix gently.
• Mix Lip2000 gently beforeuse, then dilute the appropriate amount in 50 μlofOpti-MEMI Medium (or other medium without serum). Mix gently and incubatefor 5 minutes at room temperature.
Note: Combine the dilutedLip2000 with the diluted DNA within 30 minutes. Longer incubation times maydecrease activity. If DMEM is used as a diluent for the Lip2000, mix with thediluted DNA within 5 minutes. After the 5 minute incubation,combine the diluted DNA with the dilutedLip2000 (total volume is100 μl).
•Mix gently and incubate for 20 minutes at roomtemperature to allow the DNALip2000 complexes to form. The solution may appearcloudy,but this will not inhibit the transfection.
Note:DNA-Lip2000 complexes are stable forat least 5 hours at room temperature.
2. Add the 100 μl of DNA-Lip2000 complexesto 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 readyto assay for transgene expression. It is not necessary to remove the complexesor change the medium;however,growth medium may be replaced after 4-6hours without loss of transfection activity.
For stable cell lines:Passage the cells at a 1:10 or higherdilution into fresh growth medium 24 hours after transfection. Add selective mediumthe following day.
For suspension cells:Add PMA and/or PHA (if desired) 4 hoursafter 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. ForK562 cells, adding PMA alone is sufficient to enhance promoteractivity.
Scaling Up orDown Transfections
To transfect cells in differenttissue culture formats, vary the amounts of Lip2000 , DNA, cells, and medium used in proportionto the difference in surface area (see table below). With automated, highthroughput systems, larger complexing volumes are recommendedfor transfections in 96-well plates. Note: You may perform rapid 96-well platetransfections (plate cells and transfect simultaneously) by adding a suspensionof cells directly to complexes prepared in the plate. Prepare complexes and addcells at twice the cell density as
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