The Transfection &Gene Expression Experts

Avalanche®-Everyday Transfection Reagent
Cat. No. EZT-EVDY-1

Size: 0.75 ml
1.5 mlStore at 4°C

Description
As the simplified version of our most popular and powerful Avalanche®-Omni Transfection
Reagent (Cat#: EZT-OMNI-1), Avalanche®-Everyday Transfection Reagent (Avalanche®Everyday) has the basic formulation of Avalanche®-Omni, which is a new, proprietary
formulation of lipids and polymers, thus is also an exceptionally powerful and versatile nextgeneration DNA and siRNA broad spectrum transfection reagent. Avalanche®-Everyday'stransfection efficiency is very close to that of Avalanche®-Omni, but with the prices lessthan half of Avalanche®-Omni. Best for everyday use on commonly used cells.
Features:
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•
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Broad Spectrum DNA/siRNA delivery - one transfection reagent and protocol fora variety of cells.
Transfection efficiency is close to that of Avalanche®-Omni Transfection Reagent,
but with much lower prices.
Very low Cellular Toxicity because of its bio-degradability after endocytosis.
Maintain cell density, reduce experimental biases.
Same simple protocol as that of Avalanche®-Omni Transfection Reagent: Does
not require removal of serum or culture medium and does not require washingor changing of medium after introducing the reagent/DNA complex.
Best for everyday use on commonly used cells.
High levels of recombinant protein production
Ideal for high-throughput work

BEFORE YOU START:
Important Tips for Optimal Transfection
1. Prepare high-quality plasmid DNA at 0.5–5 μg/μl in deionized water or TE buffer.
Make sure the plasmids are endotoxin-free and have A260/280 absorbance ratio of
1.8–2.0. A GFP (green fluorescent protein) plasmid can be used to determine
transfection efficiency.

Avalanche®-Everyday Transfection Reagent
2. Use Opti-MEM® I Reduced Serum Medium (Life Technologies) or regular DMEM
without serum to make Avalanche®-Everyday and nucleic acid mix (Only Opti-MEM® I
will be mentioned in the remaining parts of the protocol for simplification purposes).
Do not use NaCl2 solution or PBS.
3. Maintain the same seeding conditions between experiments. Use low-passage cells;
make sure that cells are healthy and greater than 90% viable before transfection.
4. It is important to have the cells in proliferation state and 70-90% confluence at thetime of DNA transfection.
5. Avalanche®-Everyday Transfection Reagent is extremely gentle to cells. However,transfection process will impose stress on cells, no matter what type of transfection
methods you use. The trick is to get the balance between transfection efficiency andcell viability. Increasing the number of cells plated per well or decreasing DNA andAvalanche®-Everyday amount will minimize the effect of transfection on cell growthand viability. With careful optimization, as described in page 3 and 4, this can beachieved while keeping the highest transfection efficiency.
6. Don’t use antibiotics in the culture medium during the first 24 hours of transfection.

Protocols
1 DNA Transfection
1.1 Cell Seeding
For optimal DNA transfection conditions, we recommend using cells which are 70% to 90%
confluent at the time of transfection. Typically, for experiments in 24-well plates, 50,00080,000 adherent cells are seeded per well in 0.5 ml of cell growth medium without
antibiotics 24 h prior to transfection. For the different culture formats, refer to Table 1.

EZ Biosystems™ LLC ∙ 8400 Baltimore Ave ∙ College Park ∙ MD ∙ 20740 ∙ Phone: 800 614 6133 ∙ FAX: 240 241 7046

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Avalanche®-Everyday Transfection Reagent
Table 1. Recommended number of cells to seed the day before transfection in culture medium
without antibiotics
Number ofVolume of medium
Surface area
Culture vessel
Adherent cells to seed
per well to seed
per well (cm2)(Suspension Cells)
the cells (ml)7,500-10,000
96-well
0.3
0.1(4x104)50,000-80,000
24-well
1.9
0.5(2x105)
80,000-150,000
12-well
3.81(4x105)
150,000-250,000
6-well/35 mm
9.42(8x105)
250,000-800,000
60 mm/flask 25 cm225-285(2x106)
1x106-2x106
100 mm/flask 75 cm275-78.510(6x106)
2x106-5x106
150 mm/flask 175 cm2153-175
25(1.3x107)
1.2 DNA Transfection
If this is the first time that you are using Avalanche®-Everyday on a specific type of cells,follow the following procedures and Figure 1 for optimization (The optimization procedures
are extremely important for successful transfection. Since different types of cells havedifferent sensitivity to Avalanche®-Everyday, the amount of Avalanche®-Everyday as wellas the amount of DNA needed for maximum transfection on different types of cells may
differ dramatically).
As an example, the following procedures and Figure 1 are for optimization on 24-well plate.

EZ Biosystems™ LLC ∙ 8400 Baltimore Ave ∙ College Park ∙ MD ∙ 20740 ∙ Phone: 800 614 6133 ∙ FAX: 240 241 7046

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3

Avalanche®-Everyday Transfection Reagent

1. Bring Avalanche®-Everyday andserum-free medium (OptiMEM®I)
to room temperature before
starting.
2. Add 200μl of OptiMEM®I into two
1.5ml tubes (Tube A and B).
3. Add 4.0 μg of DNA to Tube A and8.0 μg of DNA to Tube B.
4. Mix Avalanche®-Everyday prior topreparing complexes. Add 6.4 μl ofAvalanche®- Everyday to both Tube
A and Tube B containing 200μl of
different concentration of DNA
solution (20.0 μg/ml and 40.0
μg/ml respectively). Vortex for 2-3
seconds. Incubate each tube for 15
minutes at room temperature.
5. After incubation, add 3.12, 6.25,12.5, 25, 50, and 100μl of
DNA/Avalanche®- Everyday
complexes dropwise directly to thecorresponding wells of the 24-well
cell culture plate (See Figure 1).
Swirl plate gently.
6. Incubate the cells at 37°C in a CO2
incubator
7. Change growth medium 12-16
hours later.
8. Expression of reporter gene activity
should generally be assessed at 2048 hours post-transfection. GFP
expression is maximal at 40-48
hours post-transfection.

Figure 1.

After you have completed the optimization steps, choose the amount of DNA andAvalanche®-Omni that gave you the optimal balance of potency & low cytotoxicity (whichusually is the lowest dose that gave you the same high transfection efficiency as otherhigher doses did) for all of your future experiments on this specific cell type.

EZ Biosystems™ LLC ∙ 8400 Baltimore Ave ∙ College Park ∙ MD ∙ 20740 ∙ Phone: 800 614 6133 ∙ FAX: 240 241 7046

www.ezbiosystems.com

4Avalanche®-Everyday Transfection Reagent

1.3 Scale Up or Down Transfections
Use Table 2 to scale the amount of DNA/Avalanche®-Everyday for your transfection
experiment.
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Table 2. Scaling Up or Down Transfection Instruction
Culture Vessel

Multiplication factor1

96-well
48-well
24-well
12-well
6-well60-mm
10-cm
T750.17
0.50
1.00
2.00
5.00
11.05
28.95
39.47

1After determining the optimum

amount of DNA/Avalanche®-Everyday
from the optimization procedures on
the above 24-well plate, use themultiplication factor to determine theDNA and Avalanche®-Everyday amountneeded for your new plate format.

2 siRNA Transfection
2.1 Cell Seeding
For optimal siRNA transfection conditions, we recommend using cells which are 50%
confluent at the time of transfection. Typically, for experiments in 6-well plates, 100,000 to150,000 cells are seeded per well in 2 ml of growth medium without antibiotics 24 h prior totransfection. For other culture formats, refer to Table 3.
Table 3. Recommended number of cells to seed the day before transfection in culture medium
without antibiotics
Culture vessel

Number of adherent
cells to seed

Surface area per Medium per well towell (cm2)
seed the cells (ml)24-well

25,000-40,000

1.90.512-well

50,000-80,000

3.8

16-well/35 mm

100,000-150,000

9.4

2

60 mm/flask 25 cm2

200,000-500,000

25-285100 mm/flask 75 cm2

0.5x106-1x106

75-78.5

10EZ Biosystems™ LLC ∙ 8400 Baltimore Ave ∙ College Park ∙ MD ∙ 20740 ∙ Phone: 800 614 6133 ∙ FAX: 240 241 7046

www.ezbiosystems.com

Avalanche®-Everyday Transfection Reagent
2.2 siRNA Transfection
For optimal siRNA-mediated silencing, we recommend using 10 to 50 nM siRNA (finalconcentration). The following conditions are given per well of a 6-well plate. For otherculture formats, please refer to Table 4.
1. Dilute 22 to 110 pmoles siRNA (final concentration: 10 to 50 nM) into 200 μl of OptiMEM® Reduced-Serum Medium or regular high glucose DMEM without serum. Mixby vortexing.
2. Briefly vortex Avalanche®-Everyday, and add 1.0-5.0 μl into the diluted siRNA.
Immediately vortex for 10 s.
3. Incubate for 15 min at RT.
4. Add the transfection mixture drop-wise into each well.
5. Gently rock the plates back and forth and from side to side, and return the plate to
the 37°C CO2 incubator.
6. Analyze after incubating for 24 h or longer.
Table 4. siRNA transfection guidelines according to the cell culture vessel per well
Culture Vessel

siRNA(pmole)
10 nMsiRNA(pmole)
50 nM

Avalanche®Everyday (µl)

Opti-MEMor DMEM(µl))

Growthmedium(ml)FinalVolume inthe well(ml)24-well

5.5

27.5*0.2-1.050

0.50.55

12-well1155*0.4-2.0

100

11.1

22110

1.0-5.0

200

22.2

44220

2.3-11.5

400

4

4.4

121605

5.8-291100

1112.1

6-well/35 mm
60 mm/
flask 25 cm2
100 mm/flask 75 cm2* Dilute Avalanche®-Everyday 1:5 with H2O prior to application (4 µl reagent + 16 µl H2O), andthen use 5 times of the volume in the table for accurate pipetting.

EZ Biosystems™ LLC ∙ 8400 Baltimore Ave ∙ College Park ∙ MD ∙ 20740 ∙ Phone: 800 614 6133 ∙ FAX: 240 241 7046

www.ezbiosystems.com

6Avalanche®-Everyday Transfection Reagent

Intended Use:All Avalanche® Series Transfection Reagents are for research use only, not intended for any animal or
human therapeutic or diagnostic use.

Avalanche® is a trademark of EZ Biosystems™ LLC. All rights reserved.
Avalanche® series Transfection Reagents are sold to the Buyer with a limited license for Research Use
Only and is not for clinical, therapeutic or diagnostic use in humans or animals.
Avalanche® series Transfection Reagents may not be re-packaged or re-sold without written
permission from EZ Biosystems™ LLC. The buyer agrees not to attempt to reverse engineer,reconstruct, synthesize or otherwise modify EZ Biosystems™ Avalanche® products. A license from EZ
Biosystems™ LLC is required for commercial application of Avalanche® series Transfection Reagents.
For obtaining a license to use those products for commercial application, contact EZ Biosystems™ LLC
via business@ezbiosystems.com. For full terms and conditions, visit: www.ezbiosystems.com.

EZ Biosystems™ LLC ∙ 8400 Baltimore Ave ∙ College Park ∙ MD ∙ 20740 ∙ Phone: 800 614 6133 ∙ FAX: 240 241 7046

www.ezbiosystems.com

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