NPT01
NeuroPorter™ Transfection Kit
Lipid formulation for nucleic acid transfections in neuronal and glial cells
grade
for molecular biology
Quality Level
form
dried film
usage
kit sufficient for 75-200 transfections
availability
available only in USA, Canada and EU
technique(s)
transfection: suitable
storage temp.
2-8°C
Related Categories
General description
Application
- C6 glioma (human)
- Cortical neurons (rat primary)
- Dorsal Root Ganglion (DRG) cells (rat)
- NT2 neurons(human precursor cells)
- NT neurons (human differentiated cells)
- Subventricular Zone (SVZ) cells (mouse)
- White matter cells (mouse)
Features and Benefits
- Optimized for primary neurons, glial cells, and cultured neural cell lines
- Very low toxicity with no neuro-degeneration or dendrite withdrawal
- Efficient DNA delivery primary neurons, glial cells, and cultured neural cell lines
- Fast and easy to use compared to other methods
- Compatible with both serum and serum-free transfection protocols
Components
1.5 mL Hydration Buffer H9036
7.5 mL DNA Diluent D1941
Caution
Principle
Legal Information
related product
Storage Class Code
10 - Combustible liquids
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
Certificates of Analysis (COA)
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If available for a given product, the recommended re-test date or the expiration date can be found on the Certificate of Analysis.
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Why do I see a precipitate in my cell culture after lipid-based transfection?
The precipitate is likely excess lipid or EDTA and will probablly not affect transfection efficiency. If your DNA plasmid is suspended in TE, be sure the concentration of EDTA is <0.3 mM, or suspend the DNA in sterile molecular biology grade water instead.
Is low cell passage number an important consideration for transfection?
Yes, we recommend cells are at a low passage when being used for any application, including transfection. The reason why depends on what type of cells they are. Primary cells will undergo a finite number of divisions, and as they get closer to senesence they divide more slowly - both affecting their ability to take up DNA (transient transfection), and minimizing their abillity to incorporate the DNA into the genome (stable selection).Cultured common cell lines are often immortalized, and generally continue to aquire mutations, leading to a heterogenous population that may perform differently from cells of lower passage number - leading to results that are not reproducible.
Is the size of the plasmid an important consideration for transfection?
The size of the plasmid should be considered when selecting a transfection reagent with the best efficiency. In general, larger sized plasmids should easily transfect with readily available transfection reagents, as along as the plasmid DNA is of high purity.
Is optimizing the transfection protocol important?
For many common cell lines, transfection reagent efficiency is very high and the protocols will not require any optimization. For hard-to-transfect cells or those ultimately expressing a toxic protein, the protocol should be optimized for best transfection efficiency. Taking time to optimize will give you more transfected cells with each procedure, which can mean more protein expressed and results that are reproducible.
How do I choose a transfection reagent?
There are many guides that help you select a transfection reagent. In general, consider:The type of cell(s) you will transfectThe type of nucleic acid or protein you will introduce to the cellThe composition of your cell culture mediumThe need for stable or transient transfectionThe equipment you have availableThe other factors important to you - cost, protocol flexibility, ease of use, etc.
What quality does the DNA need to be in order to use it for transfection?
The DNA needs to be good quality or it may cause the cells to lyse and/or they won't transfect efficiently. Plasmid DNA prepared with a column-based DNA purification kit is suitable for transfections. Sigma's GenElute™ Minprep, Midiprep and Maxiprep kits work well for DNA plasmid purification. After preparing the DNA, confirm the OD A260:A280 ratio is greater than 1.6 for use in plasmid transfections.
What is transfection efficiency?
Transfection efficiency is a measure of how many cells take up the DNA during the transfection process. Many transfection reagents can achieve a transfection efficiency of >90% in common cell lines. Other cell lines are hard to transfect, and require special reagents and/or techniques to achieve even a small population of transfected cells.
How can I determine the efficiency of my transfection?
Calculating transfection efficiency is very useful when optimizing transfection protocols. Transfection efficiency can be performed using a GFP-expressing plasmid. After transfection, cells are stained with propidium iodide and counted. The propidium iodide provides a count of the total cells in the population, and the GFP-expressing cells provide a count of the number of cells transfected. The transfection efficiency (%) can then be calculated by:(# GFP-expressing cells / total cell #) * 100
How can I increase the efficiency of my transfection?
Transfection efficiency is affected by many different things, including plasmid size and purity, media components present, transfection reagent selected, amount of DNA and transfection reagent used, cell density, etc. Optimizing the protocol with respect to these concerns will allow you to achieve a higher transfection efficiency. For many cell lines and transfection reagents, optimized protocols are already available.
Can I transfect cells plated at low density?
For most transfections, cells should be >70% confluency the day of transfection, and growing in mid-log phase. Some transfection reagents are now designed to work with cells at low density, when required.
Can antibiotics be present in the medium during transfection?
We recommend that no antibiotics are present during transfection. The process of transfection can make the cells somewhat more porous to allow for efficient DNA entry. During this time, antibiotics will also enter the cells more easily and the cells may show increased cell death. Wait until about 24 hours after transfection to resume the use of preventative antibiotics and/or start the use of selective antibiotics.
Why are neurons difficult to transfect?
Neurons are both non-dividing cells, and sensitive to toxicity from the transfection reagent used. Sigma's Neuroporter™ Transfection Reagent is an optimized transfection reagent with a ready-to-use protocol that is helping overcome these limitations.
What is the difference between stable and transient transfection?
When the DNA enters the nucleus of the cell, the plasmid is replicated by the cell machinery (transient transfection). During this time, RNA is transcribed and protein translated until the plasmid DNA is lost after a few cell divisions. This expression of the plasmid DNA, mRNA, and protein is transient (temporary).In some cases, the plasmid DNA is integrated into the host cell genome. This is usually accompanied by forced expression using a selection antibiotic and sometimes a cloning step (to be sure all cells have the same integration site). Once the DNA is stable, the cell line can be frozen and used to express protein for many years. Clones may even be screened for those expressing the highest amount of protein.
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Articles
Transfection is the introduction of DNA, RNA, or proteins into eukaryotic cells and is used in research to study and modulate gene expression. Thus, transfection techniques and protocols serve as an analytical tool that facilitates the characterization of genetic functions, protein synthesis, cell growth and development.
This brief webinar provides an overview of what transfection is and the methods that are used to introduce DNA or RNA into eukaryotic cells.
Related Content
Browse our convenient transfection reagent selection guide to match the best reagent for your specific cell line and application needs.
Our team of scientists has experience in all areas of research including Life Science, Material Science, Chemical Synthesis, Chromatography, Analytical and many others.
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