| Product Description |
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 × 1.63**0.0252.5 × 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 × 22.4**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.
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