| Product Description |
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. |
