lipofectamine2000_man
更新时间:2023-03-29 02:42:01 阅读量: 互联网资料 文档下载
Lipofectamine? 2000
Cat. No. 11668-027 Size: 0.75 ml
Cat. No. 11668-019 Size: 1.5 ml
Cat. No. 11668-500 Size: 15 ml
Store at +4°C (do not freeze) Description
Lipofectamine? 2000 is a proprietary formulation for the transfection of nucleic acids (DNA and RNA) into eukaryotic cells providing the following advantages:?Highest transfection efficiency in many cell types and formats (e.g. 96-well).
Refer to the Cell Lines database at http://www.77cn.com.cn for a list of cell types successfully transfected.
?Nucleic acid-Lipofectamine? 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serum.
?It is not necessary to remove complexes or change/add medium after transfection, but complexes may be removed after 4-6 hours.
Important Guidelines for Transfection
?Use the procedure on page 2 to transfect cells with short interfering RNA (siRNA) or Stealth? RNAi.
?Use the procedure on page 3 to transfect cells with plasmid DNA.
?We recommend Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) to dilute Lipofectamine? 2000 and nucleic acids before complexing.
?Do not add antibiotics to media during transfection as this causes cell death. ?Maintain the same seeding conditions between experiments.
?Test serum-free media for compatibility with Lipofectamine? 2000 since some serum-free formulations (e.g. CD293, SFM II, VP-SFM) may inhibit
cationic lipid-mediated transfection.
Note:For more tips for your RNAi experiment, refer to “Seven Steps to RNAi Success”. This manual is available from http://www.77cn.com.cn/rnai or Technical Service, as are cell-type specific RNAi transfection protocols (see “RNAi protocols”.)
Quality Control
Lipofectamine?2000 is tested for absence of microbial contamination with blood agar plates, Sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of CHO-K1 cells with a reporter plasmid.
Part No.: 11668.2k.pps Rev. Date: 11 July 2006
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
For technical support, contact tech_service@http://www.77cn.com.cn.
Page 2 Stealth? RNAi or siRNA Transfection
Use this brief procedure to transfect Stealth? RNAi or siRNA into mammalian cells in a 24-well format. For other formats, see Scaling Up or Down Transfections (page 4). All amounts and volumes are given on a per well basis. Use this procedure as a starting point; optimize transfections as described in Optimizing Stealth? RNAi or siRNA Transfection, especially if you are transfecting a mammalian cell line for the first time.
1.One day before transfection, plate cells in 500 µl of growth medium without
antibiotics such that they will be 30-50% confluent at the time of transfection.
Note: Transfecting cells at a lower density allows a longer interval between transfection and assay time, and minimizes the loss of cell viability due to cell overgrowth.
2.For each transfection sample, prepare oligomer-Lipofectamine? 2000
complexes as follows:
a.Dilute 20 pmol Stealth? RNAi or siRNA oligomer in 50 µl Opti-MEM® I
Reduced Serum Medium without serum (final concentration of RNA when added to the cells is 33 nM). Mix gently.
b.Mix Lipofectamine? 2000 gently before use, then dilute 1 µl in 50 µl Opti-
MEM® I Reduced Serum Medium. Mix gently and incubate for 5 minutes at room temperature. Note: Proceed to Step c within 25 minutes.
c.After the 5-minute incubation, combine the diluted oligomer with the
diluted Lipofectamine? 2000. Mix gently and incubate for 20 minutes at
room temperature (solution may appear cloudy).
3.Add the oligomer-Lipofectamine? 2000 complexes to each well containing
cells and medium. Mix gently by rocking the plate back and forth.
Incubate the cells at 37°C in a CO2 incubator for 24-96 hours until you are ready to assay for gene knockdown. Medium may be changed after 4-6 hours. Optimizing Stealth? RNAi or siRNA Transfection
To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying RNA and Lipofectamine? 2000 concentrations. Test 10-50 pmol RNA and 0.5-1.5 µl Lipofectamine? 2000 for 24-well format. Depending on the nature of the target gene, transfecting cells at higher densities may also be considered when optimizing conditions.
Page 3 Plasmid DNA Transfection
Use the following procedure to transfect DNA into mammalian cells in a 24-well format. For other formats, see Scaling Up or Down Transfections (page 4). All amounts and volumes are given on a per well basis. Prepare complexes using a DNA (µg) to Lipofectamine? 2000 (µl) ratio of 1:2 to 1:3 for most cell lines. Transfect cells at high cell density for high efficiency, high expression levels, and to minimize cytotoxicity. Optimization may be necessary (see Optimizing Plasmid DNA Transfection, page 4).
1.Adherent cells: One day before transfection, plate 0.5-2 x 105cells in 500 µl of
growth medium without antibiotics so that cells will be 90-95% confluent at the time of transfection.
Suspension cells: Just prior to preparing complexes, plate 4-8 x 105 cells in 500 µl of growth medium without antibiotics.
2.For each transfection sample, prepare complexes as follows:
a.Dilute DNA in 50 µl of Opti-MEM® I Reduced Serum Medium without
serum (or other medium without serum). Mix gently.
b.Mix Lipofectamine? 2000 gently before use, then dilute the appropriate
amount in 50 µl of Opti-MEM® I Medium. Incubate for 5 minutes at room temperature. Note: Proceed to Step c within 25 minutes.
c.After the 5 minute incubation, combine the diluted DNA with diluted
Lipofectamine? 2000 (total volume = 100 µl). Mix gently and incubate for
20 minutes at room temperature (solution may appear cloudy). Note:
Complexes are stable for 6 hours at room temperature.
3.Add the 100 µl of complexes to each well containing cells and medium. Mix
gently by rocking the plate back and forth.
4.Incubate cells at 37°C in a CO2 incubator for 18-48 hours prior to testing for
transgene expression. Medium may be changed after 4-6 hours.
5.For stable cell lines: Passage cells at a 1:10 (or higher dilution) into fresh
growth medium 24 hours after transfection. Add selective medium (if
desired) the following day.
Page 4Optimizing Plasmid DNA Transfection
To obtain the highest transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density as well as DNA and
Lipofectamine ? 2000 concentrations. Make sure that cells are greater than 90% confluent and vary DNA (µg): Lipofectamine ? 2000 (µl) ratios from 1:0.5 to 1:5.
Scaling Up or Down Transfections
To transfect cells in different tissue culture formats, vary the amounts of
Lipofectamine ? 2000, nucleic acid, cells, and medium used in proportion to the relative surface area, as shown in the table. With automated, high-throughput systems, a complexing volume of 50 µl is recommended for transfections in 96-well plates. Note: You may perform rapid 96-well plate transfections by plating cells directly into the transfection mix. Prepare complexes in the plate and directly add cells at twice the cell density as in the basic protocol in a 100 µl volume. Cells will adhere as usual in the presence of complexes.
Shared reagents DNA transfection RNAi transfection Culture vessel
Surf. area per well 1
Vol. of plating medium Vol. of dilution medium 2 DNA Lipofect-amine ? 2000
RNA Lipofect-amine ?
2000 96-well 0.3 cm 2 100 µl 2 x 25 µl 0.2 µg 0.5 µl 5 pmol 0.25 µl 24-well 2 cm 2 500 µl 2 x 50 µl
0.8 µg
2.0 µl 20 pmol 1.0 µl 12-well 4 cm 2
1 ml
2 x 100 µl 1.6 µg 4.0 µl 40 pmol
2.0 µl
6-well 10 cm 2 2 ml 2 x 250 µl 4.0 µg 10 µl 100 pmol 5 µl 60-mm 20 cm 2 5 ml 2 x 0.5 ml 8.0 µg 20 µl 200 pmol 10 µl 10-cm 60 cm 2
15 ml
2 x 1.5 ml 24 µg
60 µl
600 pmol 30 µl
1 Surface areas may vary depending on the manufacturer.
2
Volumes of dilution medium in Step 2a & 2b of DNA or RNAi transfection protocols.
Purchaser Notification
This product is covered by one or more Limited Use Label Licenses (see the Invitrogen catalog or our web-site, http://www.77cn.com.cn). By the use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses.
Limited Use Label License No. 27: Lipofectamine ? 2000 Reagent
Limited Use Label License No. 173: Inhibition of Gene Expression by Double-Stranded RNA Limited Use Label License No. 196: Stealth ? RNAi ©2000-2006 Invitrogen Corporation. All rights reserved.
正在阅读:
二年级语文下学期期末检测考试复习专项训练冀教版含答案04-29
2014年机械工程师真题及答案04-12
基于SharePoint技术的企业信息门户建设与应用01-04
寻找我的妈妈作文700字07-12
智能卡式水表 精品 - 图文03-17
污水处理厂工程文明、安全、环保施工具体措施示范文本04-30
2018年街道工会年度工作总结模板学习04-01
职称初次认定工作流程(2014)04-03
- 1Running man探险活动策划书
- 2Running man探险活动策划书
- 3Unit 6 An old man tried to move the mountains
- 4One day a young man was standing in the middle of1
- 5Unit 3 A man from Stratford-William Shakespeare
- 6Unit 3 A man from Stratford-William Shakespeare
- 7Unit 3 A man from Stratford-William Shakespeare
- 8Unit 6 An old man tried to move the mountains.Section A
- 9神华MAN大型离心压缩机安装总结
- 10Unit 14 Five Traits of the Educated Man综合教程一
- 2009年【中考数学压轴题汇编(含解题过程)】(十)
- PEST、波特五力分析
- 教你们怎么从菜鸟级别到电脑高手
- 飞思卡尔智能车竞赛新手入门建议
- 水利工程对环境的影响及对策分析
- 自我评价中的最经典的十个“自我”
- 一种体现科学发展观的新领导力_李锡炎
- 影响中国物业管理未来命运的因素
- 红领巾心向党教案
- 关于做手抄报的作文欣赏 三年级
- 插入式涡街流量计使用说明书
- 17章 反比例函数教案全章
- 山西省忻州市河曲县事业编考试职业能力测试每日一练带答案解析(2
- 青岛李村河污水处理厂二期工程的设计与运行
- 中级维修电工计算题
- 化学工业出版社重点书推荐
- VMware5.0版本区别及功能介绍
- 国内人员个人所得税申报表(空白)
- 行政助理工作手册
- 雅思口语第三阶段问题与答案(1)
- lipofectamine
- 2000
- man