Product Description | Aladdin's UltraBio™293 Transfection Reagent is an economical, cationic liposome-based formulation, designed specifically for highly efficient transfection of adherent cells, such as HEK293, HEK293T, HEK293A, 293FT, and other 293 cell lines.UltraBio™293 Transfection Reagent provides high transfection efficiency (over 70%) on HEK293 cell lines, with good reproducibility, simple operation, and extremely low cytotoxicity, and therefore changing culture medium is unnecessary after transfection. For more information about aladdin Transfection Reagents for transfecting adherent cells, please visit our website at http
Precautions: Use high-quality DNA to achieve higher transfection efficiency. We recommend isolating plasmid DNA using ’s Plasmid Maxi Preparation Kit (D0026).Make sure that the cells are actively dividing and reach the appropriate density at the time of transfection.To transfect adherent cells other than 293 cell lines, we recommend using Lipo6000™ Transfection Reagent (, C0526) or Lipo8000™ Transfection Reagent (, C0526). To transfect suspension 293 cell lines, the Lipo293F™ Transfection Reagent (, C0518) is recommended.Antibiotic-free and serum-free culture medium or Opti-MEM® Medium is required, but not supplied in the product.Mix Lipo293™ Transfection Reagent well gently prior to each use. Vortex or centrifuge should be avoided.Seal the Lipo293™ Transfection Reagent tightly after each use to avoid long time exposure to air that 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: The following procedures are for the introduction of plasmid DNA into adherent 293 cell lines in 6-well plates. To transfect cells in other formats of cell culture vessels, please adjust the amount of Transfection Reagent proportionally as indicated in the table below.1. Seed 293 cell lines in 6-well culture plates: The day before transfection (18-24 hours in advance), seed 2-6×105 cells per well, depending on the cell type, cell size, and the growth rate of cells. Grow cells in appropriate conditions overnight to reach a cell density of 60-70% at the time of transfection.2. Before proceeding to the following transfection steps, replace per well with 2ml of fresh antibiotic culture medium containing serum. A culture medium containing both antibiotics and serum can also be used, but the presence of antibiotics may cause cytotoxicity after transfection. 3. Prepare Lipo293™ Transfection Reagent/DNA complexesa. Dilute 2.5µg of plasmid DNA in 125µl of antibiotic-free and serum-free DMEM culture medium (either high or low sugar DMEM) or Opti-MEM® Medium in a sterile tube. Mix gently. Vortex or centrifuge should be avoided.b. Dilute 5μl of Lipo293™ Transfection Reagent in 125µl of antibiotic-free and serum-free DMEM culture medium (either high or low sugar DMEM) or Opti-MEM® Medium in a sterile tube. Mix gently. Vortex or centrifuge should be avoided.c. Add the diluted DNA to the diluted transfection reagent. Mix gently. d. Incubate for 15 minutes at room temperature to allow the Lipo293™ Transfection Reagent/DNA complexes to form. The complexes are stable at room temperature for 6 hours.96-well48-well24-wel12-wel6-well6cm dish10cm dishLipo293™ Transfection Reagent0.2μl0.5μl1μl2μl5μl10μl30μlSerum-free or Opti-MEM® Medium5μl12.5μl25μl50μl125μl250μl750μlDNA100ng250ng500ng1μg2.5μg5μg15μgSerum-free or Opti-MEM® Medium5μl12.5μl25μl50μl125μl250μl750μlNote 1: The dose of Lipo293™ Transfection Reagent can be appropriately adjusted in the range of 3-12.5μl for each well of a 6-well plate. DNA dosage can be adjusted in the range of 1-4μg. A DNA (μg): Lipo293™(μl) ratio of 1:2 is generally used. The optimal transfection condition varies depending on the cell type and culture conditions and can be optimized within the range recommended above.Note 2: Prepare a master mix when transfecting multiple cell samples with the same DNA at the same amount, and then dispense the recommended amount to each well.Note 3: 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 the transfection of DNA.4. Add 250µl of Lipo293™ 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. 5. Continue to culture cells directly without changing the culture medium. After 24-48 hours of transfection, harvest cells and evaluate 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. FAQ:1. Low transfection efficiency:a. Optimize the ratio of plasmid DNA to Lipo293™ Transfection Reagent. Increase the plasmid amount if necessary.b. Use highly purified, sterile, and contaminant-free plasmid for transfection. High-purity plasmid should have an A260/A280 ratio ranging from 1.8 to 1.9. We recommend isolating plasmid DNA using Transfection Reagent/DNA complexes should be prepared in a culture medium without antibiotics and serum.d. Make sure that the cells to be transfected are in good condition. The cell density should be 60-70 % at the time of transfection. The optimal cell density for transfection should be determined for different cell types.e. Insufficient incubation time after transfection may result in low transfection efficiency. The recommended incubation time after transfection to yield high expression of target proteins is 24-48 hours.f. Regularly check cells for mycoplasma infection. Mycoplasma contamination detrimentally affects cell proliferation, and thereby the transformation efficiency.g. If the expression of the target protein is not detected, check the sequence of the plasmid to ensure the open reading frame is correct.2. Cellular toxicity occurs:a. Plat cells at least 18-24 hours in advance prior to transfection.b. To determine if the expressed target protein is toxic to cells, cells transfected with empty expression plasmid can be used as a negative control. If the target protein is indeed toxic to cells, reduction of plasmid amount as well as Lipo293™ Transfection Reagent amount accordingly can be considered. c. Check if the cell density is too low at the time of transfection.d. Check cell culture if there is any infection of mycoplasma or other microorganisms.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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