The Transfection &Gene Expression Experts

Avalanche®-Omni Transfection Reagent
Cat. No. EZT-OMNI-1

Size: 0.75 ml
1.5 mlStore at 4°C

Description
Avalanche®-Omni Transfection Reagent is a proprietary lipid-polymer mixture that forms
lipopolyplexes with nucleic acids. It is an exceptionally powerful and versatile nextgeneration DNA and siRNA transfection reagent for day-to-day experiments. It has beenshown to effectively transfect the broadest spectrum of adherent and suspension cells. No
other transfection reagents can match the efficiency, broad spectrum capabilities,
convenience, and gentleness of Avalanche®-Omni Transfection Reagent for transfection ofprimary cells and other hard-to-transfect cells. Because of the high transfection efficiency,much lesser amounts of DNA and Avalanche®-Omni are needed to achieve high transfection
efficiency as compared to most other transfection reagents. It is best to use the smallest
amounts of DNA and transfection reagent possible in order to maintain good cell growth andviability, and generate physiologically relevant data you can trust.
Features:•••••••••

Exceptional transfection efficiency in the broadest range of cell types including
difficult-to-transfect cell lines and primary cells with super high levels of gene
expression or knockdown.
Economical: High efficiency means smaller amounts of the nucleic acid and reagent
are needed. 1.5 ml of Avalanche®-Omni Reagent is sufficient for about 3,500
transfections in 24-well plates, or about 700 transfections using 6-well plates!
Extremely gentle to cells: The chemical features of our proprietary formula and thesmaller amounts needed for transfection ensure very low toxicity levels.
Simplest procedure: – simply mix with nucleic acid and add to cell culture.
Superior performance for co-transfection of siRNA and plasmid DNA
Proven efficacy in the presence of serum – eliminates the need to change media
following transfection
100% animal origin- free
Reliable performance for high-throughput applications
The best choice for establishing stable cell lines

Avalanche®-Omni Transfection Reagent

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.
2. Use Opti-MEM® I Reduced Serum Medium (Life Technologies) or regular DMEM
without serum to make Avalanche®-Omni 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®-Omni 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 Avalanche®Omni and DNA amount will minimize the effect of transfection on cell growth andviability. With careful optimization, as described in page 3 and 4, this can be achieved
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®-Omni 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
1.90.5(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®-Omni on a specific type of cells, follow
the following procedures and Figure 1 for optimization (The optimization procedures areextremely important for successful transfection. Since different types of cells havedifferent sensitivity to Avalanche®-Omni, the amount of Avalanche®-Omni as well as theamount 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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Avalanche®-Omni Transfection Reagent

1. Bring Avalanche®-Omni and serumfree 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®-Omni prior to
preparing complexes. Add 6.4 μl ofAvalanche® -Omni 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®-Omni 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 and
Avalanche®-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®-Omni Transfection Reagent

1.3 Scale Up or Down Transfections
Use Table 2 to scale the amount of DNA/Avalanche®-Omni for your transfection experiment.

Table 2. Scaling Up or Down Transfection Instruction
Culture Vessel

Multiplication factor1

96-well
48-well
24-well
12-well
6-well
60-mm
10-cm
T75

0.17
0.50
1.002.00
5.00
11.05
28.95
39.47

51After determining the optimum

amount of DNA/Avalanche®-Omni fromthe optimization procedures on theabove 24-well plate, use themultiplication factor to determine theDNA and Avalanche®-Omni 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 to
150,000 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 well (cm2)

Medium per well toseed the cells (ml)24-well

25,000-40,000

1.9

0.5

12-well

50,000-80,000

3.8

16-well/35 mm

100,000-150,000

9.4

260 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®-Omni 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®I. Mix by vortexing.
2. Briefly vortex Avalanche®-Omni, 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
Avalanche®Omni (µl)

Opti-MEMor DMEM(µl))

Growthmedium(ml)FinalVolume inthe well(ml)27.5*0.2-1.050

0.50.551155*0.4-2.010011.1

22110

1.0-5.0

200

2

2.2

44220

2.3-11.5

400

4

4.4

121

605

5.8-291100

1112.1

Culture Vessel

siRNA(pmole)
10 nM

siRNA(pmole)
50 nM

24-well

5.512-well
6-well/35 mm
60 mm/
flask 25 cm2
100 mm/flask 75 cm2* Dilute Avalanche®-Omni 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

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Avalanche®-Omni 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 the 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 writtenpermission 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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