Storage Condition:
4°C, do not freeze
Shelf life:
Transfection Incubation Time: The transfection reaction, i.e. membrane crossing of plasmid DNA, takes place
within the first 1-4 hours. During this time, the cells should be grown in serum-free media, if possible, for maximum ef-ficienc
y
. 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 (com-
plete media) after the initial transfection period.
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
Allele’s LineSilence
TM
and Silen
-
Circle
TM
can down-regulate specific gene expression for 1-2 weeks without drug-resistance selection.
AvantGene
TM
Transfection Reagent
Description:
AvantGene
TM
2 is a broad range transfection reagent that ef-ficiently delivers DN
A molecules into a variety of cultured cells. A
vantGene has been extensively tested by
Allele scientists
in Hela, 293, 293
T
, 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
express-
ing plasmids, Allele’
s RNAi LineSilence
TM
cassettes and Allele’s
RNAi SilenCircle
TM
plasmids have been tested with AvantGene reagent.
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% confl
u
-
ence at the time of transfection. Cells are normally seeded at a lower density
, e.g. 20-40%, approximately 24 hours before
transfection. A
vantGene Reagent:
A
vantGene reagent has low cell toxicity and can work in a wide range of concentrations. For a 24-well plate transfection, use 2.5
µ
l AvantGene reagent as a starting point. In general, more A
vantGene reagent within a 2-3 fold
range from the starting point results in higher transfection ef
-ficienc
y, however, higher amount of reagent might also result in
toxicity in certain cells. Refer to
T
able 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
T
able 1 for recommended starting conditions.
Please See Reverse Side For Recommended UseBox 1 | Catalog Numbers
ABP-TC-
A
TR125U
125 µl
ABP-TC-
A
TR250U
250 µl
ABP-TC-
A
TR750U
750 µl 6 months
Protocol for Transfection in 24-well plate:
1.
Plate cells approximately 24 hours prior to transfection at a cell density of 20-40% confluence in complete media (with serum and antibiotics if required) normally used for the cell type being used.
2.
Mix 2.5 µl AvantGene reagent to 10 µl serum free, antibiotic-free media, incubate 5-10min at room temperature.
3.
Add 12.5 µl DNA Diluent to 0.5 µg 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 µl 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 µl 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 adher-ent cells. While waiting for the incubation, spin down the sus-pension 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 10
6
cells per ml.
3.
Prepare the DNA/AvantGene 2 mixture as above, add di-rectly to the suspension cells, gently pipette up and down 2-3 times. Proceed as for attached cells.
Culture Plate:6-well12-well24-well96-wellSurface Area:9.4 cm23.8 cm21.9 cm20.32 cm2 AvantGene:5-20 μl2.5-10 μl1.25-5 μl0.5-1.25 μlSerum-free media:200 μl100 μl50 μl10 μlComplete media:800 μl400 μl200 μl100 μlPlasmid DNA:1-4 μg0.5-2 μg0.25-1 μg0.1-0.25 μg All volumes in Table 1 are for one well on indicated plate.Table 1. Starting conditions for using AvantGene
Note: The volume of DNA Diluent should be proportional to the amount of DNA, e.g. 25 µl Diluent for 1 µg DNA; The volume of serum-free medium used to dilute the transfection reagent should be protortional to the amount of AvantGene
TM
2 used, e.g. 20 l medium for 5 µl reagent.
Satisfaites votre curiosité
Tout ce que vous voulez lire.
À tout moment. Partout. Sur n'importe quel appareil.
Aucun engagement. Annulez à tout moment.