UltraBio™8000转染试剂(UltraBio™8000 Transfection Reagent)是阿拉丁最新研发的一种以纳米材料为基础的最便捷的高效细胞转染试剂，达到甚至超过了国际主流转染试剂的转染效果，并且转染过程无须任何孵育时间，实现了细胞转染的至简操作。适用于把质粒、siRNA或其它形式的核酸包括DNA、RNA、寡核苷酸、以及核酸蛋白复合物或带负电荷的蛋白转染到真核细胞中，也可以用于活体动物的核酸转染以及用于基因治疗。UltraBio™8000转染试剂的纳米技术可保证其瞬时转染和稳定转染时的可靠性和稳定性。UltraBio™8000转染试剂使用特别便捷，无血清培养液和核酸及转染试剂可以直接混匀，质粒转染无需任何孵育时间，即可直接加入到细胞培养器皿内，实现了细胞转染的至简操作。UltraBio™8000转染试剂对于常见的哺乳动物细胞具有非常高的转染效率、重复性好、操作简单、通常观察不到细胞毒性，对于贴壁细胞和悬浮细胞都适用，特别适用于难转染的贴壁细胞。UltraBio™8000转染试剂由于几乎完全没有细胞毒性，从而在转染后无需进行细胞培养液的更换，在转染24-48小时后直接收集细胞进行蛋白表达鉴定即可。UltraBio™8000转染试剂经过对小鼠胚胎成纤维细胞NIH3T3、人胚胎肾细胞HEK293/HEK293T、宫颈癌细胞Hela、结肠癌细胞HCT116、肝细胞HepG2和肺癌细胞A549等多种细胞的测试，转染效率和Thermo公司的UltraBio™3000Transfection Reagent、Promega公司的ViaFect Transfection Reagent相当，在一些细胞中甚至比UltraBio™ 3000和ViaFect的转染效率更高。贴壁细胞转染试剂的比较和选择请参考：http://www.aladdin-e.com。UltraBio™8000转染试剂不仅适用于质粒、siRNA等单一成分的细胞转染，也适合多个质粒或者质粒与siRNA等的组合转染。UltraBio™8000转染试剂转染过表达质粒后，通常24-48小时后达到较高的蛋白表达水平，并且很多情况下蛋白表达量在转染后48小时显著高于转染后24小时；转染siRNA通常3-5天后对于目的基因的下调水平会比较理想。UltraBio™8000转染试剂转染细胞时，不受培养液中的血清和抗生素的影响，即可以在血清和抗生素存在的情况下进行细胞转染。UltraBio™8000转染试剂的转染效果可以通过转染表达EGFP等荧光蛋白的质粒进行快速鉴定。UltraBio™8000转染试剂与UltraBio™ 3000 Reagent转染效果比较请参考图1-8。每毫升本转染试剂大约可以转染10厘米培养皿40个、6厘米培养皿125个、6孔板250个孔、12孔板625个孔、24孔板1250个孔、48孔板2500个孔、96孔板6250个孔。

注意事项：

使用说明：

Aladdin's UltraBio™8000 Transfection Reagent is a highly efficient and easy-to-use reagent for nanomaterial-mediated nucleic acid delivery. It demonstrates comparable or superior transfection performance to other commercially available transfection reagents. It is versatile for delivery of siRNA or other forms of nucleic acids including DNA, RNA, oligonucleotides, and nucleic acid protein complexes or negatively charged proteins into eukaryotic cells, as well as for nucleic acid transfection of live animals and gene therapy. The nanoparticle technology of the UltraBio™8000 Transfection Reagent ensures the reliability and stability of both transient and stable transfections of cells.The UltraBio™8000 Transfection Reagent is extremely easy to use. Cell transfection can be achieved by mixing the UltraBio™8000 Transfection Reagent directly with the serum-free culture medium and nucleic acid. Cell transfection with plasmid even does not need incubation, which minimize the operation time significantly. The UltraBio™8000 Transfection Reagent has been tested on mouse embryonic fibroblast cells NIH3T3, human embryonic kidney cells HEK293/HEK293T, cervical cancer cells Hela, colon cancer cells HCT116, hepatocytes HepG2 and lung cancer cells A549. The UltraBio™8000 Transfection Reagent yield high transfection efficiency comparable to UltraBio™ 3000 Transfection Reagent and Promega's ViaFect™ Transfection Reagent. In some cells it delivers more efficiently than UltraBio™ 3000 and ViaFect™ transfection. For more comparison and selection of transfection reagents for adherent cells, please visit http

Precautions：

Transfection efficiency may decrease with the increase of Lipo8000™ Transfection Reagent. Start with the recommended dosage in the protocol. Optimize the dosage only when necessary.Use high quality DNA or RNA to achieve a higher transfection efficiency. Plasmid Mass Extraction Kit (D0026)from can be used for plasmid purification to ensure a high transfection efficiency.Cells must be in an optimum physiological condition for transfection.Changing medium 4-6 hours after transfection does not interfere with the transfection efficiency. If necessary, culture medium can be changed 4-6 hours after transfection.Antibiotic-free and serum-free culture medium, such as Opti-MEM® culture medium or DMEM culture medium, is required but not supplied in this product. Mix the Lipo8000™ Transfection Reagent gently. Vortex or centrifuge should be avoided.Close the Lipo8000™ tube tightly to avoid the exposure of the transfection reagent to air, which may reduce the transfection efficiency.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear lab coats and disposable gloves while handling kit reagents .

Instructions for Use：

1.DNA transfection(General protocol for transfection of adherent and suspension cells in a 6-well plate): a.The day before transfection (18-24 hours), seed about 200-700,000 cells per well in 6-well plates, depending on the cell type, size and the growth rate of cells. At the time of transfection, the optimal confluency for adherent cells is 70-80%. Suspension cells should be in logarithmic growth phase at the time of transfection. b.Prior to the following transfection steps, replace with 2ml of fresh culture medium (complete medium containing serum and antibiotics) per well. The presence of antibiotics does not affect the transfection efficiency of Lipo8000™ Transfection Reagent, nor cause cytotoxicity after transfection.c.Preparation of Lipo8000™ Transfection Reagent/DNA complexe: For each well of a 6-well plate, mix 2.5µg of plasmid DNA gently by pipetting with 125µl of antibiotic-free and serum-free DMEM culture medium (either high or low sugar DMEM) or Opti-MEM® Medium in a sterile eppendorf tube, followed by adding 4µl of Lipo8000™ Transfection Reagent. Mix well gently by pipetting. Vortex or centrifuge should be avoided. The Lipo8000™ Transfection Reagent/DNA complexes are stable for up to 6 hours at room temperature. 96-well48-well24-well12-well6-well6cm dish10cm dishSerum-free culture medium or Opti-MEM® Medium5μl12.5μl25μl50μl125μl250μl750μlDNA100ng250ng500ng1μg2.5μg5μg15μgLipo8000™ Transfection Reagent0.16μl0.4μl0.8μl1.6μl4μl8μl24μlMix DNA with medium gently first, and then add Lipo8000™ Transfection Reagent. Mix well gently. Incubation is not necessary. Note 1: The dose of Lipo8000™ Transfection Reagent can be appropriately adjusted in the range of 2-6μl for each well of a 6-well plate. DNA dosage is recommended to be fixed at 2.5µg, but can be adjusted in the range of 1-4µg. A DNA (μg): Lipo8000™ (μl) ratio of 1:2 or 1:3 is generally used, but can be ranging from 1:0.5 to 1:5 for optimal transfection efficiency. The ratio in the above table is 1:2 with a lower dosage of Lipo8000™, which is both economical and efficient. The optimal transfection condition varies depending on the cell type and culture conditions, and can be optimized within the range recommended above.Note 2: The concentration of plasmids should be in the range of 0.5-5µg/μl.Note 3: A mastermix can be prepared when transfecting multiple cell samples with same plasmids at same amount, and then distribute the recommended volume to each well.Note 4: For other formats of cell culture vessels, the dose of each reagent can be scaled up or down accordingly based on the culture area of cell culture vessels. For transfection of oligonucleotides or RNA, please refer to the protocol for DNA transfection.d.Add 125µl of Lipo8000™ Transfection Reagent/DNA complexes to each well. Gently rock the plate to ensure an even distribution of the complexes in the well, and continue to culture directly without changing the culture medium.e.Incubate at 37℃in a CO2 incubator for 24-48 hours. Harvest cells and examine the transfection efficiency by appropriate methods such as fluorescence assay, Western Blot, ELISA, reporter gene etc. To obtain cells with stable transfection, grow cells in selective medium with antibiotics such as G418.2.siRNA transfection:a.The day before before transfection (18-24 hours), seed about 200-700,000 cells per well in 6-well plates, depending on the cell type, size and the growth rate of cells. At the time of transfection, the optimal confluency for adherent cells is 70-80% . Suspension cells should be in logarithmic growth phase at the time of transfection. b.Prior to the following transfection steps, replace wells with 2ml of fresh culture medium (complete medium containing serum and antibiotics) per well. The presence of antibiotics does not affect the transfection efficiency of Lipo8000™ Transfection Reagent or cause cytotoxicity after transfection.c.For each well of a 6-well plate, mix 100pmol of siRNA gently by pipetting with 125µl of antibiotic-free and serum-free DMEM culture medium (either high or low sugar DMEM) or Opti-MEM® Medium in a sterile eppendorf tube, followed by adding 4µl of Lipo8000™ Transfection Reagent. Mix well gently by pipetting and incubate at room temperature for 20 minutes prior to transfection. Vortex or centrifuge should be avoided. The Lipo8000™ Transfection Reagent/siRNA complexes are stable for up to 6 hours at room temperature.96-well48-well24-well12-well6-well6cm dish10cm dishSerum-free culture medium or Opti-MEM® Medium5μl12.5μl25μl50μl125μl250μl750μlsiRNA4pmol10pmol20pmol140pmol100pmol200pmol600pmolLipo8000™ Transfection Reagents0.16μl0.4μl0.8μl1.6μl4μl8μl24μlMix gently after adding siRNA, add Lipo8000™ Transfection Reagent and incubate for 20 minutes at room temperature. Note 1: The dose of Lipo8000™ Transfection Reagent can be appropriately adjusted in the range of 2-6μl for each well of a 6-well plate. siRNA dosage can be adjusted in the range of 50-250pmol. A siRNA (pmol): Lipo8000™(μl) ratio of 25:1 is generally used, but can be ranging from 10:1 to 40:1 for optimal transfection efficiency. The ratio in the above table is 25:1 with a lower dosage of Lipo8000™, which is both economical and efficient. The optimal transfection condition varies depending on the cell type and culture conditions, and can be optimized within the range recommended above.Note 2: The recommended concentration of siRNA is 20µM. A siRNA concentration ranging from 10µM to 50µM is generally used.Note 3: A mastermix can be prepared when transfecting multiple cell samples with same siRNA at same amount, and then distribute the recommended volume to each well.Note 4: For other formats of cell culture vessels, the dose of each reagent can be scaled up or down accordingly based on the culture area of cell culture vessels. For transfection of oligonucleotides or RNA, please refer to the protocol for transfection of DNA.d.Add 125μl of Lipo8000™ Transfection Reagent/siRNA complexes to each well. Gently shake the plate to ensure an even distribution of the complexes in the well, and continue to culture directly without changing the culture medium.e.Incubate at 37℃ in a CO2 incubator for more than 48 hours. Analyze the expression of target gene by appropriate methods such as qPCR, Western Blot, ELISA, reporter gene etc. For genes with a long half-life of mRNA, a significant decrease in mRNA or protein levels might be detected 3-5 days after transfection with siRNA or miRNA.FAQ:1.Low transfection efficiency:a.Optimize the ratio of plasmid to Lipo8000™ Transfection Reagent. Increase the amount of Lipo8000™ Transfection Reagent for cells that are difficult-to-transfect.b.Use highly purified, sterile and contaminant-free plasmids for transfection. High purity DNA should have a A260/A280 ratio ranging from 1.8 to 1.9. Transfection Reagent might be toxic to some cells with low cell density.b.Use same amount of DNA or siRNA, but reduce the amount of Lipo8000™ Transfection Reagent by 25%. For example, instead of 2.5µg of plasmid and 4µl of Lipo8000™ used regularly, mix 2.5µg of plasmid and 3µl of Lipo8000™ for each well of a six-well plate to avoid cytotoxicity, which has no obvious effect on transfection efficiency.c.Reduce the amount of plasmids and also Lipo8000™ Transfection Reagent proportionally. If not working, reduce the amount of Lipo8000™ Transfection Reagent further by 25% .d.Make sure cell density is not too low at the time of transfection. Adherent cells should be 70-80 % confluent and suspension cells should be in logarithmic growth phase at the time of transfection.e.Some cells are sensitive to Lipo8000™ Transfection Reagent. Replace the medium containing transfection reagent with new growth medium 4-6 hours post-transfection, which does not affect transformation efficiency.f.Check cells for mycoplasma or other microorganism infection.Appendix:Data sheets relating to the size, culture area, cell culture volume and recommended culture volume of commonly used multi-well plates and petri dishes:Multiple Well Plates or DishesSingle Well Only for PlatesDiameter (Bottom, mm)*Growth Area(cm2)*Average Cell YieldTotal Well Volume (ml)WorkingVolume (ml)Recommended Volume (ml)6 well34.89.59.5 × 10516.81.9-2.9212 well22.13.83.8 × 1056.90.76-1.14124 well15.61.91.9 × 1053.40.38-0.570.548 well11.00.959.5 × 1041.60.19-0.2850.2596 well6.40.323.2 × 1040.360.10-0.200.1384 well2.70.0565.6 × 1030.1120.025-0.0500.0301536 well1.63 × 1.63**0.0252.5 × 1030.01250.005-0.0100.0103.5 cm dish3499.0 × 105NA1.8-2.726 cm dish52212.1 × 106NA4.2-6.3510 cm dish8.4555.5 × 106NA11-16.51215cm dish141521.5 × 107NA30.4-45.63524.5cm dish22.4 × 22.4**5005.0 × 107NA100-150120*Diameter and growth area may vary depending on the manufacturer, and the listed sizes are from Corning. **These wells or dishes are square.