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Transfection Reagent Troubleshooting

How do you compare Avalanche™-Omni Transfection Reagent with other commercial transfection reagents?

Avalanche™-Omni Transfection Reagent is an exceptionally powerful and versatile next-generation DNA and siRNA transfection reagent for day-to-day experiments. It has been shown to effectively transfect the broadest spectrum of adherent and suspension cells. No other transfection reagents can match the efficiency, spectrum, convenience, and gentleness of Avalanche™-Omni Transfection Reagent for transfection of primary, challenging, and sensitive cell lines. It includes a streamlined, simple protocol where the complexes are added directly to cells without changing media. This lends itself to high throughput applications. It works very well in the presence or absence of serum.

Is it important to do a double CsCl purification of plasmid DNA prior to transfection? Is it possible to use column-based purification methods?

DNA needs to be pure enough to be exposed to the cells otherwise they will lyse, or the transfection efficiency will be so low as to be ineffective. Both CsCl purification and column-based purification methods should produce high quality DNA. After any purification, be sure to remove all the ethanol before resuspending the DNA in sterile water or TE. It is highly recommended to do a "cells + DNA only" control to check for any adverse side effects of the DNA on the cells. Additionally, leave one plate with just cells and media as a control. 

How can I optimize transfection conditions for Avalanche™-Omni Transfection Reagent?

Optimization of transfection conditions is essential for the highest-efficiency transfections and the lowest toxicity. The conditions that should be optimized include Avalanche™-Omni Transfection Reagent and DNA concentrations, the ratio of them, and cell number etc. To optimize the amount of Avalanche™-Omni Transfection Reagent, start with cells at 80-90% confluency and 0.5 µg DNA for 24-well plates. With cell number and DNA concentration held constant, vary the amount of Avalanche-Omni™ to determine the optimal concentration. The product protocol gives a simple procedure for optimization. The cell number and amount of DNA can also be optimized. It is possible to minimize the effect of transfection on cell growth and viability by increasing the number of cells plated per well or by decreasing either Avalanche-Omni™ Transfection Reagent or DNA concentrations. With careful optimization this can be achieved with little impact on the level of transgene expression.

Can antibiotics be used in media during transfection?

We discourage using any antibiotics during transfection (e.g. Geneticin®, Hygromycin, Gentamycin, Penicillin, etc). There can be higher cell death when antibiotics are present during transfection. Even though some such as penicillin and streptomycin are not toxic to eukaryotic cells in a healthy culture, during transfection the cell permeability increases so that much higher levels of antibiotics get into cells. For stable transfections, wait at least 24-48 h after transfection before adding selective antibiotics.

How do I choose from Avalanche™ Transfection Reagents series?

It depends on your transfection goals. If you want to have a broad spectrum transfection reagent that provides excellent transfection efficiency on most of your cell types for day-to-day experiments with minimum optimization, Avalanche-OmniTransfection Reagent (EZT-OMNI-1) is the one that you need. If you want to achieve the best transfection result for a specific type of cells, use cell type-specific Avalanche™ transfection reagents. Your first step is to go to our product list, and find out the cell type/cell line specific Avalanche™ transfection reagents for your cells by identifying the respective cell types or cell lines. Use the Filters on the left to narrow down the product list. The cell type/cell line specific Avalanche™ transfection reagents were specifically designed for transfection on the respective cell types or cell lines. The proprietary formulations ensures the highest possible transfection efficiencies and viabilities without comprehensive optimization. Click HERE to find out how EZ Biosystems developed the cell type/cell line specific Avalanche™ transfection reagents.

How can I get the maximum transfection efficiency with Avalanche™-Omni Transfection Reagent?

  • Optimize the Reagent and DNA amounts. The most important parameter is the ratio of the Reagent to DNA. 
  • Do not use serum during complex formation. Serum contains negatively charged proteins which will interfere with complex formation. Opti-MEM® I Reduced-Serun Medium or DMEM (use either medium in the absence of serum during complex formation) is good media for complex formation.  Do not use NaCl solution to make complexes. 
  • Do not use antibiotics, EDTA, citrate, phosphate, RPMI, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare the DNA-Reagent complexes. 
  • Cell density should be from 80% to 90% confluent at the time of transfection. Cells should be in the mid-log growth phase. 
  • Make sure the promoter-enhancer of the transfected DNA is compatible with the target cell type. 
  • Include a positive control for the transfection assay. 

Is the passage number of my cells important to consider when doing transfection?

In general, once optimal transfection conditions are determined for a given cell line, it is recommended that cells be passaged less than 20 times to maintain reproducible results. Thus immediately following the determination of optimal conditions, cells should be frozen down so that when the working stock approaches 20 passages, a new batch can be started from the frozen stock.

What is the best Complex formation medium:

Opti-MEM® Reduced-Serum Medium  (Cat# 31985-070, Life Technologies) or regular DMEM without serum.

Is it necessary to use serum-free media during transfection?

Not in all cases. What is essential is to form the reagent:nucleic acid complex in the absence of serum, because negatively charged proteins can interfere with complex formation.  Once the complexes are formed, they can be added to cells in serum containing medium.

Will nicked DNA lead to reduced transfection efficiency?

Yes. You should verify the integrity of your DNA on an agarose gel to see if it is degraded. Supercoiled plasmid runs faster than linear plasmid. Nicked plasmid will run slower than linear plasmid.
The content of nicked DNA in your DNA preparation should be below 20%. Higher content of nicked DNA results in significant decrease of transfection efficiency.

How stable are  the Avalanche-Omni™ Transfection Reagents?

All products of the Avalanche™ Transfection Reagent series are stable at 4º C for at least one year. Do not freeze.

Do I really need to include a control when I doing siRNA transfection?

It is absolutely critical to have a control oligonucleotide to be able to determine any non-specific effects. This oligonucleotide can be a scrambled oligonucleotide (same length and base composition in a random order) or a sense oligonucleotide if the target is mRNA.

Will low A260/A280 ratio lead to both reduced transfection efficiency and cell viability?

Yes. To check the quality of your DNA, we strongly recommend determining the A260:A280 ratio. It should be at least 1.7 for a good DNA preparation.

Can siRNA and plasmid be co-transfected into cells?

Yes,  the general procedure below is recommended to cotransfect your plasmid DNA and an RNAi molecule into mammalian cells using Avalanche™ Transfection Reagents. Detailed cotransfection methods are included in the product protocols.

  • One day before transfection, plate cells in the appropriate amount of growth medium without antibiotics such that they will be 80-90% confluent at the time of transfection.
  • For each transfection sample, prepare DNA-RNAi molecule-Avalanche™ Transfection Reagents complexes as follows: 
    • Dilute the DNA and RNAi molecule in the appropriate amount of Opti-MEM® I Medium without serum. Mix gently.
    • Mix Avalanche™ Transfection Reagents  gently before use. Add respective amount of Avalanche™ Transfection Reagent into the diluted DNA and RNAi molecule . Immediately mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur. 
  • Add the DNA-RNAi molecule-Avalanche™ Transfection Reagents complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth. 
  • Incubate the cells at 37 °C in a CO2 incubator until you are ready to harvest cells and assay for your target gene. Removal of complexes or media change is not required; however, growth medium may be replaced after 4-6 hours without loss of transfection activity.
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