阿拉丁L266177

Lipo2000 Transfection Reagent

产品说明

Lipo2000转染效率介于Lip2000和Lip3000之间，毒性更低，转染效率更高，是一种新型的阳离子脂质体转染试剂。适合于将核酸(DNA和RNA)转染入真核细胞，具有低细胞毒性；对多种类型的细胞和培养板都具有高转染效率；转染时血清的存在不影响转染效率的优点。

适用范围：贴壁细胞和悬浮细胞（哺乳动物细胞系）的转染。

质粒DNA的转染

对大多数细胞来说，DNA(µg)与Lipo2000 (µl)的比例为1:2~1:3。转染时高的细胞密度可以得到高的转染效率和表达水平，并能减少细胞毒性。

1. 以24孔板为例

贴壁细胞： 转染前一天，用500 µl不含抗生素的培养基接种0.5～2×10^5细胞，使之第二天能达到70-90%汇合。

悬浮细胞：在准备DNA-Lip2000复合物之前，用500 µl不含抗生素的培养基接种4～8×10^5细胞即可。

2. 对每个转染样品，进行以下操作

a. 在eppendorf管里分别加入50 µl Opti-MEM I ReLipced Serum Medium和0.8 µg DNA轻柔混匀，制成DNA稀释液。

b. 在另一个eppendorf管里分别加入50µl Opti-MEMI ReLipced Serum Medium和2.0 µl Lipo2000(注意用前先混匀)，轻柔混匀，制成Lip2000 稀释液，室温静置5分钟。

c. 将DNA稀释液和Lip2000稀释液混合，轻柔混匀，室温静置20分钟， 形成DNA-Lip2000复合物。DNA-Lip2000复合物在室温下可稳定存在6小时。

3. 将DNA-Lip2000复合物加入到接种好的细胞中，将培养板轻轻地前后摇动，使复合物分散均匀。

4. 在37℃ CO2培养箱中培养4-6小时后更换培养基,继续培养18～48小时。

5. 如果要筛选稳定细胞株，则在转染24小时后将细胞按照1:10或更高的比例接种到新鲜培养基中，第二天加入选择性培养基进行筛选。

质粒DNA转染的优化 为达到最高的转染效率和降低细胞毒性的影响，可以对DNA和Lip2000的比例以及细胞密度进行优化，一般在1:0.5~1:5的范围内优化DNA (µg)和Lip2000 (µl) 的比例。

常见细胞的转染效率（仅供参考，实验条件不同转染效率会有差别）

Product manual

The transfection efficiency of Lipo2000 is between Lip2000 and Lip3000, with lower toxicity and higher transfection efficiency. It is a new type of cationic liposome transfection reagent. Suitable for transfection of nucleic acids (DNA and RNA) into eukaryotic cells, with low cytotoxicity; high transfection efficiency for various types of cells and culture plates; the presence of serum during transfection does not affect the advantages of transfection efficiency .

Scope of application: transfection of adherent cells and suspension cells (mammalian cell lines).

Transfection of plasmid DNA

For most cells, the ratio of DNA (µg) to Lipo2000 (µl) is 1:2~1:3. High cell density during transfection can obtain high transfection efficiency and expression level, and can reduce cytotoxicity.

1. Take 24-well plate as an example

Adherent cells: On the day before transfection, inoculate 0.5~2×10^5 cells with 500 µl of antibiotic-free medium to reach 70-90% confluence the next day.

Suspension cells: Before preparing the DNA-Lip2000 complex, inoculate 4-8×10^5 cells with 500 µl of antibiotic-free medium.

2. Perform the following operations for each transfection sample

a. Add 50 µl Opti-MEM I ReLipced Serum Medium and 0.8 µg DNA into the eppendorf tube and mix gently to make a DNA dilution.

b. Add 50 µl Opti-MEMI ReLipced Serum Medium and 2.0 µl Lipo2000 to another eppendorf tube (note that you should mix well before use), mix gently to prepare a Lip2000 dilution, and let it stand at room temperature for 5 minutes.

c. Mix the DNA diluent and Lip2000 diluent, mix gently, and let stand at room temperature for 20 minutes to form a DNA-Lip2000 complex. The DNA-Lip2000 complex can exist stably for 6 hours at room temperature.

3. Add the DNA-Lip2000 complex to the inoculated cells, and gently shake the culture plate back and forth to make the complex evenly dispersed.

4. After culturing in a 37°C CO2 incubator for 4-6 hours, change the medium and continue culturing for 18 to 48 hours.

5. If you want to screen for stable cell lines, inoculate the cells in a fresh medium at a ratio of 1:10 or higher 24 hours after transfection, and add selective medium for screening the next day.

Optimization of plasmid DNA transfection In order to achieve the highest transfection efficiency and reduce the impact of cytotoxicity, the ratio of DNA to Lip2000 and cell density can be optimized. Generally, DNA (µg) is optimized in the range of 1:0.5~1:5 And Lip2000 (µl) ratio.

The amount of media, nucleic acid and Lipo2000 for transfection in different cell culture plates

Transfection efficiency of common cells (for reference only, the transfection efficiency will vary with different experimental conditions)