The calcium phosphate transfection method is performed differently than other methods, and so it can be optimized beyond the general recommendations. An important part of the protocol is generation of a fine precipitate. Be sure that the bubbling in tube B is consistant and with good force, while the solution from tube A is added very slowly, dropwise. Once the precipitates are added to the cell culture, be sure they are evenly distributed on the plate so all cells may be transfected.The pH of the HeBS is critical to precipitate formation. Prolonged storage of the solution may cause the pH to shift away from the optimal 7.05 - 7.12.
CAPHOS
Calcium Phosphate Transfection Kit
Most cost effective transfection reagent kit for transient and stable transfection of DNA into mammalian cells
Synonym(s):
Gene delivery
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€550.00
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1 KIT
€550.00
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€550.00
In StockDetails
grade
Molecular Biology
Quality Level
form
solution
usage
kit sufficient for 160 transfections (6 cm dishes)
kit sufficient for 400 transfections (3.5 cm dishes)
kit sufficient for 80 transfections (10 cm dishes)
technique(s)
transfection: suitable
shipped in
dry ice
storage temp.
−20°C
1 of 4
This Item | NPT01 | GALS | 72181 |
|---|---|---|---|
| technique(s) transfection: suitable | technique(s) transfection: suitable | technique(s) microbe id | staining: suitable | technique(s) transfection: suitable |
| usage kit sufficient for 160 transfections (6 cm dishes), kit sufficient for 80 transfections (10 cm dishes), kit sufficient for 400 transfections (3.5 cm dishes) | usage kit sufficient for 75-200 transfections | usage kit sufficient for 100 tests (using a 3.5 cm dish) | usage - |
| Quality Level 200 | Quality Level 200 | Quality Level 200 | Quality Level 200 |
| shipped in dry ice | shipped in - | shipped in dry ice | shipped in wet ice |
| storage temp. −20°C | storage temp. 2-8°C | storage temp. −20°C | storage temp. 2-8°C |
| grade for molecular biology | grade for molecular biology | grade - | grade - |
General description
Calcium phosphate transfection is a commonly used method for the introduction of DNA into eukaryotic cells. This technique has been used to obtain both transient and stable transfections in a wide variety of cell types.
Application
Calcium Phosphate Transfection Kit has been used:
Suitable for transient and stable transfection of DNA into cultured mammalian cells. The following cells have successfully been transfected using the calcium phosphate method:
BAEC
Bowels melanoma cells
CHO K1
COS-7
Fibroblasts (human embryonic, neo derm)
HEK293
Huh 7
IMR-90
LLC (Lewis Lung Carcinoma)
NIH3T3
PC-12
PCI-13
SH-Sy5Y
SK-Hep-1
T47D
BAEC
Bowels melanoma cells
CHO K1
COS-7
Fibroblasts (human embryonic, neo derm)
HEK293
Huh 7
IMR-90
LLC (Lewis Lung Carcinoma)
NIH3T3
PC-12
PCI-13
SH-Sy5Y
SK-Hep-1
T47D
Features and Benefits
- Suitable for transient and stable transfection
- Reproducible for a wide range of cell types
- Widely referenced
- Inexpensive
Components
The Calcium Phosphate Transfection Kit contains:
5 ml 2.5M CaCl2 (C2052)
25 ml 2x HEPES Buffered Saline (H1012)
25 ml molecular biology grade water (W4502)
5 ml 2.5M CaCl2 (C2052)
25 ml 2x HEPES Buffered Saline (H1012)
25 ml molecular biology grade water (W4502)
Principle
The procedure is based on slow mixing of HEPES-buffered saline containing sodium phosphate with a CaCl2 solution containing the DNA. A DNA-calcium phosphate co-precipitate forms, which adheres to the cell surface and is taken up by the cell, presumably by endocytosis.
Other Notes
The protocol was developed for transient transfection of CHO cells using pSV40-CAT plasmid as a reporter gene.
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Warning
Hazard Statements
Precautionary Statements
Hazard Classifications
Eye Irrit. 2
Storage Class Code
12 - Non Combustible Liquids
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
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How can I increase the efficiency of the calcium phosphate transfection method?
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Is optimizing the transfection protocol important?
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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.
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How can I determine the efficiency of my transfection?
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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
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How can I increase the efficiency of my transfection?
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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.
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What is transfection efficiency?
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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.
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Is low cell passage number an important consideration for transfection?
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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.
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What is the difference between stable and transient transfection?
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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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What quality does the DNA need to be in order to use it for transfection?
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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.
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How do I choose a transfection reagent?
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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.
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Why do I see a precipitate in my cell culture after calcium-phosphate transfection?
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The precipitate is normal to see - this is the calcium/DNA precipitate the cell will internalize during the transfection process. If bubbled correctly, the precipitates will be very fine and regular sized, then evenly distributed over the cells.
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