转染试剂

Lipofectamine™ RNAiMAX

Cat. No. 13778-075 Size: 0.75 ml

Cat. No. 13778-150 Size: 1.5 ml

Store at +4°C (do not freeze) Description

Lipofectamine™ RNAiMAX is a proprietary formulation specifically developed for the transfection of siRNA and Stealth™ RNAi duplexes into eukaryotic cells. Lipofectamine™ RNAiMAX provides the following advantages:

• High transfection efficiencies in many cell types to minimize background expression from untransfected cells and maximize knockdown.

• Minimal cytotoxicity to reduce non-specific effects and cellular stress.

• Generally requires low concentrations of RNAi duplexes to obtain high knockdown levels, further minimizing non-specific effects.

• A broad peak of optimal transfection activity with minimal cytotoxicity, allowing achievement of high knockdown levels despite differences in cell density, minor pipetting inaccuracies, and other variations.

Important Guidelines for Transfection

• Reverse transfection (page 2) and forward transfection (page 3) protocols can be used for most cell lines tested. Cell-type specific transfection protocols are available at http://www.77cn.com.cn/RNAi or through Technical Service.

• We recommend Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) to dilute RNAi duplexes and Lipofectamine™ RNAiMAX before complexing.• Do not add antibiotics to media during transfection as this causes cell death.• Test serum-free media for compatibility with Lipofectamine™ RNAiMAX. • To assess transfection efficiency, we recommend using a KIF11 Stealth™Select RNAi, as described in Assessing Transfection Efficiency (page 2). • Use 10 nM RNAi duplex and indicated procedure as a starting point;

optimize transfections as described in Optimizing Transfections (page 3). Quality Control

Lipofectamine™ RNAiMAX is tested for absence of microbial contamination with blood agar plates, Sabaraud dextrose agar plates, and fluid thioglycolate medium, for absence of RNAse activity, and functionally by transfection of Stealth™ RNAi and appropriate controls into a reporter cell line.

Part No.: 13778.PPS Rev. Date: 11 Jan 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.

转染试剂

Page 2 Reverse Transfection

Use this procedure to reverse transfect Stealth™ RNAi or siRNA into mammalian cells in a 24-well format (for other formats, see Scaling Up or Down Transfections, page 4). In reverse transfections, the complexes are prepared inside the wells, after which cells and medium are added. Reverse transfections are faster to perform than forward transfections, and are the method of choice for high-throughput transfection. Optimize transfections as described in Optimizing Transfections (page 3), especially if transfecting a mammalian cell line for the first time. All amounts and volumes are given on a per well basis.

1. For each well to be transfected, prepare RNAi duplex-Lipofectamine™

RNAiMAX complexes as follows.

a. Dilute 6 pmol RNAi duplex in 100 µl Opti-MEM® I Medium without serum

in the well of the tissue culture plate. Mix gently.

b. Mix Lipofectamine™ RNAiMAX gently before use, then add 1 µl

Lipofectamine™ RNAiMAX to each well containing the diluted RNAi

molecules. Mix gently and incubate for 10-20 minutes at room

temperature.

2. Dilute cells in complete growth medium without antibiotics so that 500 µl

contains the appropriate number of cells to give 30-50% confluence 24 hours after plating. Use 20,000-50,000 cells/well for suspension cells.

3. To each well with RNAi duplex - Lipofectamine™ RNAiMAX complexes, add

500 µl of the diluted cells. This gives a final volume of 600 µl and a final RNA concentration of 10 nM. Mix gently by rocking the plate back and forth.

4. Incubate the cells 24-72 hours at 37°C in a CO2 incubator until you are ready

to assay for gene knockdown.

Assessing Transfection Efficiency

To qualitatively assess transfection efficiency, we recommend using a KIF11 Stealth™ Select RNAi (available through http://www.77cn.com.cn/rnaiexpress; for human cells, oligo HSS105842 is a good choice). Adherent cells in which

KIF11/Eg5 is knocked down exhibit a “rounded-up” phenotype after 24 hours due to a mitotic arrest (Weil, D. et al., Biotechniques (2002), 33: 1244-1248); slow growing cells may take up to 72 hours to display the rounded phenotype. Alternatively, growth inhibition can be assayed after 48-72 hours.

Note: The BLOCK-iT™ Fluorescent Oligo (Cat. No. 2013) is optimized for use with Lipofectamine™ 2000, and is not recommended for Lipofectamine™ RNAiMAX.

转染试剂

Page 3 Forward Transfection

Use this procedure to forward transfect Stealth™ RNAi or siRNA into mammalian cells in a 24-well format (for other formats, see Scaling Up or Down Transfections, page 4). In forward transfections, cells are plated in the wells, and the transfection mix is generally prepared and added the next day. Optimize transfections as described in Optimizing Transfections (page 3), especially if transfecting a mammalian cell line for the first time. All amounts and volumes are given on a per well basis.

Note: For some cell lines (e.g. MCF-7 or HepG2), we recommend reverse transfections.

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.

2. For each well to be transfected, prepare RNAi duplex-Lipofectamine™

RNAiMAX complexes as follows:

a. Dilute 6 pmol RNAi duplex in 50 µl Opti-MEM®I Reduced Serum Medium …… 此处隐藏：6761字，全部文档内容请下载后查看。喜欢就下载吧 ……