基本描述

稳定性与储存	4℃保存。长期不使用可以-20℃保存。
英文名称	UltraBio™6000 Transfection Reagent
储存温度	-20°C储存
运输条件	超低温冰袋运输
产品介绍	产品介绍： UltraBio™6000转染试剂(UltraBio™6000 Transfection Reagent)是一种非常高效的新型转染试剂，达到了国际最主流转染试剂的转染效果。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用于基因治疗。UltraBio™6000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、无明显的细胞毒性，并且对于贴壁细胞和悬浮细胞都适用。贴壁细胞转染试剂的比较和选择请参考：http://www.aladdin-e.com。UltraBio™6000转染试剂的使用方法和常用的UltraBio™2000Reagent基本一致。并且经过对HEK293T、Hela、NIH3T3、HEK293FT、CHO等细胞的测试，转染效率也和UltraBio™2000Reagent相当甚至略高。UltraBio™6000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™6000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™6000转染试剂转染细胞时，基本不受细胞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。但为了取得最佳的转染效果，推荐转染时使用不含抗生素的含血清的细胞培养液。UltraBio™6000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™6000转染试剂与UltraBio™2000Reagent转染效果比较请参考图1-6。对于六孔板，一个包装的C0526-0.5ml、C0526-1.5ml和C0526-7.5ml转染试剂大约可以分别转染100个、300个和1500个孔；对于24孔板，一个包装的C0526-0.5ml、C0526-1.5ml和C0526-7.5ml转染试剂大约可以分别转染500个、1500个和7500个孔。注意事项：使用高纯度的DNA或RNA有助于获得较高的转染效率。对于质粒，可以使用阿拉丁生产的质粒大量抽提试剂盒进行抽提，以保证可以获得较高的转染效率。转染前细胞必须处于良好的生长状态。需自备不含抗生素的无血清培养液或Opti-MEM?培养液或普通的DMEM培养液。Lipo6000?转染试剂不能vortex或离心，宜缓慢晃动混匀。Lipo6000?转染试剂使用后请立即盖好盖子，避免长时间暴露在空气中，影响转染效率。本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。为了您的安全和健康，请穿实验服并戴一次性手套操作。\t'], 使用说明： 1.DNA转染：a.细胞培养(以六孔板为例，其它培养板或培养皿可参考六孔板)：在转染前一天(18-24小时)按照每孔约20-70万细胞(具体的细胞数量据细胞类型、大小和细胞生长速度等而定)接种到六孔板内进行培养，使第二天细胞密度能达到约70-90%。b.在进行下述转染步骤前，把培养有细胞的六孔板每孔换成2ml新鲜培养液(含有血清，不含抗生素)。可以使用含有血清并含有抗生素的新鲜培养液，但抗生素的存在对于有些细胞容易导致转染后出现一定的细胞毒性。c.参考下表，对于待转染的六孔板中每一个孔的细胞，取两个洁净无菌离心管，分别加入125μl不含抗生素和血清的DMEM培养液(高糖DMEM或低糖DMEM均可)或Opti-MEM® Medium，然后于其中一管加入2.5μg质粒DNA，并用枪轻轻吹打混匀；另一管加入5μl Lipo6000™转染试剂，用枪轻轻吹打混匀，请特别注意不可Vortex或离心。室温静置5分钟后(通常最长不宜超过25分钟)，将含有DNA的培养液用枪轻轻加入含Lipo6000™转染试剂的培养液中，轻轻颠倒离心管或者用枪轻轻吹打混匀，室温静置5分钟(室温存放6小时内稳定)。96-well48-well24-well12-well6-well 6cm dish10cm dishLipo6000™转染试剂0.2μl 0.5μl 1μl 2μl 5μl 10μl 30μl 无血清培养液或Opti-MEM® Medium5μl 12.5μl 25μl 50μl 125μl 250μl 750μl DNA100ng 250ng 500ng 1μg 2.5μg 5μg 15μg 无血清培养液或Opti-MEM® Medium5μl 12.5μl 25μl 50μl 125μl 250μl 750μl 稀释好的Lipo6000™转染试剂和DNA分别室温静止放置5分钟，随后两者混合并混匀再室温静止放置5分钟每孔加入的混合物的量10μl 25μl 50μl 100μl 250μl 500μl 1500μl 按照上述用量每孔均匀滴加Lipo6000™转染试剂和DNA的混合物，4-6小时后更换培养液或直接继续培养注1：对于六孔板中一个孔的细胞，Lipo6000™转染试剂的用量可以在3-12.5μl范围内进行适当调节，DNA用量建议固定在2.5μg，但也可以在1-4μg的范围内进行适当调节。通常质粒用量(μg)和Lipo6000™(μl)的用量比例为1:2或1:3比较常用，如有必要可以在1:0.5-1:5的范围内优化转染效果，上表推荐的比例为1:2，此时Lipo6000™的用量相对较少，既经济又高效。最佳的转染条件，因不同的细胞类型和培养条件而有所不同，可以在上述推荐范围内自行优化转染条件。注2：质粒的浓度宜控制在0.5-5μg/μl范围内。注3：对于多个孔转染相同数量相同质粒的情况可以把每个孔所需的Lipo6000™转染试剂和DNA混合物分别配制，然后一起混合在同一个离心管内，后续混匀并孵育5分钟后，可以按照推荐用量滴加到细胞培养器皿内。注4：对于其它培养板或培养器皿，各种试剂的用量可以按照细胞培养器皿的培养面积按比例进行换算。如果转染寡核苷酸或RNA等可以参考转染DNA的条件进行。d.无论是贴壁细胞还是悬浮细胞，按照六孔板每孔250μl Lipo6000™转染试剂-DNA混合物的用量，均匀滴加到整个孔内，随后轻轻混匀。e.为达到最高的转染效率，细胞在转染后培养4-6小时后宜更换为新鲜的完全培养液(对于Hela细胞，推荐在转染4小时后更换培养液，对于NIH3T3、CHO、HEK293T和HEK293FT细胞，推荐在转染6小时后更换培养液)。f.继续培养约24-48小时后，即可用适当方式检测转染效果，例如荧光检测、Western、ELISA、报告基因等，或加入适当的筛选药物如G418等进行稳定细胞株的筛选。2.siRNA转染：a.细胞培养(以六孔板为例，其它培养板或培养皿可参考六孔板)：在转染前一天(18-24小时)按照每孔约20-70万细胞(具体的细胞数量据细胞类型、大小和细胞生长速度等而定)接种到六孔板内进行培养，使第二天细胞密度能达到约30-50%。b.在进行下述转染步骤前，把培养有细胞的六孔板每孔换成2ml新鲜培养液(含有血清，不含抗生素)。可以使用含有血清并含有抗生素的新鲜培养液，但抗生素的存在对于有些细胞容易导致转染后出现一定的细胞毒性。c.参考下表，对于待转染的六孔板中每一个孔的细胞，取两个洁净无菌离心管，分别加入125μl不含抗生素和血清的DMEM培养液(高糖DMEM或低糖DMEM均可)或Opti-MEM® Medium，然后于其中一管加入100pmol siRNA，并用枪轻轻吹打混匀；而另一管加入5μl Lipo6000™转染试剂，用枪轻轻吹打混匀，请特别注意不可Vortex或离心。室温静置5分钟后(通常最长不宜超过25分钟)，将含有siRNA的培养液用枪轻轻加入含Lipo6000™转染试剂的培养液中，轻轻颠倒离心管或者用枪轻轻吹打混匀，室温静置5分钟(室温存放6小时内稳定)。96-well48-well24-well12-well6-well 6cm dish10cm dishLipo6000™转染试剂0.2μl 0.5μl 1μl 2μl 5μl 10μl 30μl 无血清培养液或Opti-MEM® Medium5μl 12.5μl 25μl 50μl 125μl 250μl 750μl siRNA4pmol10pmol20pmol40pmol100pmol 200pmol600pmol无血清培养液或Opti-MEM® Medium5μl 12.5μl 25μl 50μl 125μl 250μl 750μl 稀释好的Lipo6000™转染试剂和siRNA分别室温静止放置5分钟，随后两者混合并混匀再室温静止放置5分钟每孔加入的混合物的量10μl 25μl 50μl 100μl 250μl 500μl 1500μl 按照上述用量每孔均匀滴加Lipo6000™转染试剂和siRNA的混合物，4-6小时后更换培养液或直接继续培养注1：对于六孔板中一个孔的细胞，Lipo6000™转染试剂的用量可以在2.5-7.5μl范围内进行适当调节，siRNA用量可以在50-250pmol的范围内进行适当调节。通常siRNA用量(pmol)和Lipo6000™(μl)的用量比例为20:1，如有必要可以在10:1-40:1的范围内优化转染效果，上表推荐的比例为20:1，此时Lipo6000™的用量相对较少，既经济又高效。最佳的转染条件，因不同的细胞类型和培养条件而有所不同，可以在上述推荐范围内自行优化转染条件。注2：siRNA的推荐浓度为20μM，常用的浓度范围为10-50μM。注3：对于多个孔转染相同数量相同质粒的情况可以把每个孔所需的Lipo6000™转染试剂和siRNA混合物分别配制，然后一起混合在同一个离心管内，后续混匀并孵育5分钟后，可以按照推荐用量滴加到细胞培养器皿内。注4：对于其它培养板或培养器皿，各种试剂的用量可以按照细胞培养器皿的培养面积按比例进行换算。如果转染寡核苷酸或RNA等可以参考转染DNA的条件进行。d.无论是贴壁细胞还是悬浮细胞，按照六孔板每孔250μl Lipo6000™转染试剂-siRNA混合物的用量，均匀滴加到整个孔内，随后轻轻混匀。e.为达到最高的转染效率，细胞在转染后培养4-6小时后宜更换为新鲜的完全培养液(对于Hela细胞，推荐在转染4小时后更换培养液，对于NIH3T3、CHO、HEK293T和HEK293FT细胞，推荐在转染6小时后更换培养液)。f.继续培养3-5天后，即可用适当方式检测siRNA对于靶基因的下调效果，例如qPCR、Western、ELISA、报告基因等。常见问题：1.转染效率低：a.优化质粒与Lipo6000™转染试剂比例，对于难转染的细胞，可适当加大质粒用量。b.应用高纯度、无菌、无污染物的质粒进行转染，DNA纯度方面A260/A280比值要接近1.8，通常宜控制在1.8-1.9范围内，偏低则有可能有蛋白污染，偏高则有可能有RNA污染。可以使用阿拉丁生产的质粒大量抽提试剂盒进行抽提，以保证可以获得较高的转染效率。c.贴壁细胞转染时状态良好，细胞密度达30-50%时才可进行转染，过稀可能影响转染效率，细胞密度达到50-90%时通常不会影响转染效率。不同细胞的最佳转染密度需要自行摸索。悬浮细胞宜在对数生长期进行转染。d.需使用无抗生素和无血清培养液配制Lipo6000™转染试剂和质粒或siRNA等的混合物。e.转染后培养时间不足，而被误以为转染效率偏低。不同细胞转染后至显著表达所需要培养的时间通常为24-48小时。f.检查细胞是否有支原体感染，支原体感染会影响细胞增殖，并很可能影响转染效率。g.如果没有检测到目的蛋白表达，应该仔细核对转染质粒的测序结果，确保测序结果和读码框完全正确。h.如果靶基因的敲减(knockdown)效果欠佳，应该考虑尝试设计不同的siRNA。2.细胞毒性较大：a.缩短转染时间，在转染后较短时间内更换新鲜的细胞培养液。b.减少质粒用量，按照比例减少Lipo6000™转染试剂。c.检查是否转染时细胞密度太低。附录：常用多孔板和培养皿的尺寸、培养面积、细胞培养量和推荐的培养体积等相关数据表：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 × 105 16.81.9-2.9212 well22.13.83.8 × 105 6.90.76-1.14124 well15.61.91.9 × 105 3.40.38-0.570.548 well11.00.959.5 × 104 1.60.19-0.2850.2596 well6.40.323.2 × 104 0.360.10-0.200.1384 well2.70.0565.6 × 103 0.1120.025-0.0500.0301536 well1.63 × 1.630.0252.5 × 103 0.01250.005-0.0100.0103.5 cm dish3499.0 × 105 NA1.8-2.726 cm dish52212.1 × 106 NA4.2-6.3510 cm dish8.4555.5 × 106 NA11-16.51215cm dish141521.5 × 107 NA30.4-45.63524.5cm dish22.4 × 22.45005.0 × 107 NA100-150120Diameter and growth area may vary depending on the manufacturer, and the listed sizes are from Corning. These wells or dishes are square. Aladdin's UltraBio™8000 Transfection Reagent is a novel and highly efficient transfection reagent for nucleic acid delivery. It demonstrates comparable or superior transfection performance to other commercially available transfection reagents. It is applicable in introducing plasmids, 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 in nucleic acid transfection of live animals and gene therapy.UltraBio™6000 Transfection Reagent provides high transfection efficiency, good reproducibility, and simple operation in transfecting common mammalian cells without obvious cytotoxicity, and is applicable to both adherent and suspension cells. For more comparison and selection of transfection reagents for adherent cells, please refer to the following webpage Precautions：* Use high-quality DNA to achieve higher transfection efficiency. We recommend isolating plasmid DNA using ’s Plasmid Maxi Preparation Kit .Cells must be in an optimum physiological condition for 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 Lipo6000™ Transfection Reagent gently. Vortex or centrifuge should be avoided.Close the Lipo6000™ tube tightly after each use to avoid 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 a lab coat and disposable gloves during the operation. Instructions for Use： 1. DNA transfection:The following procedures are for the transfection of cells in a 6-well plate. To transfect cells in other formats of cell culture vessels, adjust the reagent volume proportionally as indicated in the table below.a. The day before transfection (18-24 hours), seed 200-700,000 cells per well in 6-well plates, depending on the cell type, size, and growth rate of cells. At the time of transfection, the optimal confluency for adherent cells is 70-80%. Suspension cells should be in the 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, but no antibiotics) per well. A complete medium containing both serum and antibiotics can also be used, but the presence of antibiotics may cause cytotoxicity of some cells after transfection.c. For each well to be transfected, take two sterile tubes and add 125µl of antibiotic-free and serum-free DMEM culture medium (either high or low sugar DMEM) or Opti-MEM® Medium into each tube. Add 2.5µg plasmid DNA into one of those two tubes and 5µl of Lipo6000™ Transfection Reagent into the other. Mix well gently by pipetting (Avoid vortex or centrifuge). After incubation at room temperature for 5 minutes (no more than 25 minutes), combine the diluted plasmid and the diluted Transfection Reagent, mix well by pipetting up and down gently, and incubate again at room temperature for 5 minutes. The Lipo6000™ Transfection Reagent/DNA complexes are stable for up to 6 hours at room temperature.96-well48-well24-well12-well6-well6cm dish10cm dishLipo6000™ Transfection Reagent0.2μl0.5μl1μl2μl5μl10μl30μlSerum-free culture medium or Opti-MEM® Medium5μl12.5μl25μl50μl125μl250μl750μlDNA100ng250ng500ng1μg2.5μg5μg15μgSerum-free culture medium or Opti-MEM® Medium5μl12.5μl25μl50μl125μl250μl750μlIncubate the diluted DNA and the diluted Lipo6000™ Transfection Reagent at room temperature for 5 minutes. Mix two dilutions gently and then incubate again at room temperature for 5 minutes.Note 1: The dose of Lipo6000™ Transfection Reagent can be appropriately adjusted in the range of 3-12.5μl 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): Lipo6000™ (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: Prepare a master mix when transfecting multiple cell samples with the same plasmids at the same amount, and then dispense the recommended amount 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 the transfection of oligonucleotides or RNA, please refer to the protocol for DNA transfection.d. Add 250µl of Lipo6000™ Transfection Reagent/DNA complexes drop-wise to each well of a 6-well plate. Gently shake the culture plate to evenly distribute the transfection complexes.e. To achieve the best transfection efficiency, replace the transfection mixture with fresh complete medium 4-6 hours after transfection. For Hella cells, change medium 4 hours after transfection. For NIH3T3, CHO, HEK293T, and HEK293FT cells, change medium 6 hours after transfection. f. Incubate at 37℃in a CO2 incubator for 24-48 hours. Harvest cells and analyze 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 a selective medium with antibiotics such as G418.2. siRNA transfection: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 growth rate of cells. At the time of transfection, the optimal confluency for adherent cells is 30-50%. Suspension cells should be in the 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, but no antibiotics) per well. A complete medium containing both serum and antibiotics can also be used, but the presence of antibiotics may cause cytotoxicity of some cells after transfection.c. For each well to be transfected, take two sterile tubes and add 125µl of antibiotic-free and serum-free DMEM culture medium (either high or low sugar DMEM) or Opti-MEM® Medium into each tube. Add 100pmol of siRNA into one of those two tubes and 5µl of Lipo6000™ Transfection Reagent into the other. Mix well gently by pipetting (Avoid vortex or centrifuge). After incubation at room temperature for 5 minutes (no more than 25 minutes), combine the diluted siRNA and the diluted Transfection Reagent, mix well by pipetting up and down gently, and incubate again at room temperature for 5 minutes. The Lipo6000™ Transfection Reagent/siRNA complexes are stable for up to 6 hours at room temperature.96-well48-well24-well12-well6-well6cm dish10cm dishLipo6000™ Transfection Reagent0.2μl0.5μl1μl2μl5μl10μl30μlSerum-free culture medium or Opti-MEM® Medium5μl12.5μl25μl50μl125μl250μl750μlsiRNA4pmol10pmol20pmol40pmol100pmol200pmol600pmolSerum-free culture medium or Opti-MEM® Medium5μl12.5μl25μl50μl125μl250μl750μlIncubate the diluted siRNA and the diluted Lipo6000™ Transfection Reagent at room temperature for 5 minutes. Mix two dilutions gently and then incubate again at room temperature for 5 minutes.Note 1: The dose of Lipo6000™ Transfection Reagent can be appropriately adjusted in the range of 2.5-7.5μl 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 20:1 is generally used, but can be ranging from 10:1 to 40:1 for optimal transfection efficiency. The ratio in the above table is 20:1 with a lower dosage of Lipo6000™, 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: Prepare a master mix when transfecting multiple cell samples with the same plasmids at the same amount, and then dispense the recommended amount 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 the transfection of oligonucleotides or RNA, please refer to the protocol for DNA transfection.d. Add 250µl of Lipo6000™ Transfection Reagent/siRNA complexes drop-wise to each well of a 6-well plate. Gently shake the culture plate to evenly distribute the transfection complexes.e. To achieve the best transfection efficiency, replace the transfection mixture with fresh complete medium 4-6 hours after transfection. For Hella cells, change medium 4 hours after transfection. For NIH3T3, CHO, HEK293T, and HEK293FT cells, change medium 6 hours after transfection. f. Incubate at 37℃ in a CO2 incubator for 3-5 days. Analyze the expression of the target gene by appropriate methods such as qPCR, Western Blot, ELISA, reporter gene, etc.FAQ:1. Low transfection efficiency:a. Optimize the ratio of plasmid to Lipo6000™ Transfection Reagent. Increase the amount of plasmids for cells that are difficult to transfect.b. Use highly purified, sterile, and contaminant-free plasmids for transfection. High-purity DNA should have an A260/A280 ratio ranging from 1.8 to 1.9. Transfection Reagent/plasmid or siRNA complexes should be prepared in a culture medium without antibiotics and serum.e. Insufficient incubation time after transfection may result in low transfection efficiency. The incubation time required for significant expression in different cells after transfection is usually 24-48 hours.f. Regularly check cells for mycoplasma infection. Mycoplasma contamination detrimentally affects cell proliferation, and thereby the transfection efficiency.g. If the expression of the target protein is not detected, check the sequence of the transfected plasmids to ensure that its open reading frame is correct.h. If the knockdown of target genes is not effective, design a new siRNA sequence.2. Cellular toxicity occurs：a. Decrease the transfection time and replace a fresh cell culture medium within a relatively short time after transfection.b. Reduce the amount of plasmid and also Lipo6000™ Transfection Reagent proportionally. c. Check if the cell density is too low at the time of transfection.Appendix: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)AverageCell YieldTotal WellVolume (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× 105 3.40.38-0.570.548 well11.00.959.5× 104 1.60.19-0.2850.2596 well6.40.323.2 × 1040.360.10-0.200.1384 well2.70.0565.6 × 1030.1120.025-0.0500.0301536 well1.63 × 1.630.0252.5 × 1030.1250.005-0.0100.0103.5 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 × 22.45005.0 × 107NA100-150120Diameter and growth area may vary depending on the manufacturer, and the listed sizes are from Corning. *These wells or dishes are square.

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