AvantGene Transfection Reagent

Box 1 | Catalog Numbers

ABP-TC-ATR125U ABP-TC-ATR250U ABP-TC-ATR750U 125 l 250 l 750 l

TM

Storage Condition: Shelf life:

4C, do not freeze 6 months

Description:
AvantGeneTM2 is a broad range transfection reagent that efciently delivers DNA molecules into a variety of cultured cells. AvantGene has been extensively tested by Allele scientists in Hela, 293, 293T, CHO, COS-7, HUVEC, 3T3-L1, NIH 3T3, MCF-7, Vero E6, C2C12 and rat L6 muscle cells and found to be superior to many other transfection reagents. cDNA expressing plasmids, Alleles RNAi LineSilenceTM cassettes and Alleles RNAi SilenCircleTM plasmids have been tested with AvantGene reagent. Transfection Incubation Time: The transfection reaction, i.e. membrane crossing of plasmid DNA, takes place within the rst 1-4 hours. During this time, the cells should be grown in serum-free media, if possible, for maximum efciency. If the experimental cells can not grow or attach in serum-free media, AvantGene may be used in the presence of serum. Antibiotics should be avoided during the incubation, but may be added back with serum-containing media (complete media) after the initial transfection period.

General Considerations for Transfection:

Cell Density: Continued cell division after transfection often helps transfected DNA to enter the nucleus where transcription occurs. The recommended cell density is about 50-70% conuence at the time of transfection. Cells are normally seeded at a lower density, e.g. 20-40%, approximately 24 hours before transfection. AvantGene Reagent: AvantGene reagent has low cell toxicity and can work in a wide range of concentrations. For a 24-well plate transfection, use 2.5 l AvantGene reagent as a starting point. In general, more AvantGene reagent within a 2-3 fold range from the starting point results in higher transfection efciency, however, higher amount of reagent might also result in toxicity in certain cells. Refer to Table 1 for recommended conditions for transfection of different scales. DNA Purity and Concentration: DNA used for transfection should be of high purity and free from endotoxin. The amount or concentration of DNA may need to be optimized for each cell type. Refer to Table 1 for recommended starting conditions.

Transfection of GFP plasmid into rat muscle cells using AvantGene

Observation Period: Transfected cells typically show cDNA expression starting around 8-12 hours and last for a few days. DNA-based RNAi such as Alleles LineSilenceTM and SilenCircleTM can down-regulate specic gene expression for 1-2 weeks without drug-resistance selection.

Please See Reverse Side For Recommended Use

Table 1. Starting conditions for using AvantGene Culture Plate: Surface Area: AvantGene: Serum-free media: Complete media: Plasmid DNA: 6-well 9.4 cm2 5-20 l 200 l 800 l 1-4 g 12-well 3.8 cm2 2.5-10 l 100 l 400 l 0.5-2 g 24-well 1.9 cm2 1.25-5 l 50 l 200 l 0.25-1 g 96-well 0.32 cm2 0.5-1.25 l 10 l 100 l 0.1-0.25 g

All volumes in Table 1 are for one well on indicated plate.

Protocol for Transfection in 24-well plate:
1. Plate cells approximately 24 hours prior to transfection at a cell density of 20-40% conuence in complete media (with serum and antibiotics if required) normally used for the cell type being used. 2. Mix 2.5 l AvantGene reagent to 10 l serum free, antibioticfree media, incubate 5-10min at room temperature. 3. Add 12.5 l DNA Diluent to 0.5 g DNA, incubate 1-5 min at room temperature. Do not incubate longer than 5 min. 4. During the waiting time in step 3, change the media of the cells to be transfected to 200 l serum-free and antibiotic-free medium. 5. Mix diluted transfection reagent from Step 2 to DNA solution from Step 3, incubate at room temperature for 5-10 min. Do not incubate longer than 30 min. 6. Add the transfection mixture from step 5 drop-wise to the well. 7. After 2-4 hours incubation under appropriate conditions in an incubator, add 250 l serum-containing normal medium.

Protocol for Transfection of Suspension Cells:

1. Split the cells the day before transfection so that they are in optimal growth condition on the day of transfection. 2. Prepare DNA and AvantGene 2 mixes as above for adherent cells. While waiting for the incubation, spin down the suspension cells, and resuspend them in serum-free medium (or serum-containing medium if the cells get sick without serum during transfection). Use the volume according to the above table. The cell density should be around 106 cells per ml. 3. Prepare the DNA/AvantGene 2 mixture as above, add directly to the suspension cells, gently pipette up and down 2-3 times. Proceed as for attached cells.

Note: The volume of DNA Diluent should be proportional to the amount of DNA, e.g. 25 l Diluent for 1 g DNA; The volume of serum-free medium used to dilute the transfection reagent should be protortional to the amount of AvantGeneTM2 used, e.g. 20 l medium for 5 l reagent.