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.
Fortfahren mit
CAPHOS
Calciumphosphat-Transfektionskit
Most cost effective transfection reagent kit for transient and stable transfection of DNA into mammalian cells
Synonym(e):
Gene delivery
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| Größe/SKU | Verfügbarkeit | Preis |
|---|---|---|
1 kit | Warenkorb auf Verfügbarkeit prüfen | CHF 582.00 |
Über diesen Artikel
UNSPSC Code:
12352200
EC Number:
233-140-8
NACRES:
NA.85
eCl@ss:
38110301
Form:
solution
Grade:
Molecular Biology
Technique(s):
transfection: suitable
CHF 582.00
Warenkorb auf Verfügbarkeit prüfen
Technischer Dienst
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Unterstützung erhaltengrade
Molecular Biology
Quality Segment
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
General description
Die Calciumphosphat-Transfektion ist eine häufig genutzt Methode für das Einführen von DNA in eukaryotische Zellen. Diese Methode wird verwendet, und in einer großen Vielfalt von Zellarten sowohl transiente als auch stabile Transfektionen zu erzielen.
Application
Das Calciumphosphat-Transfektionskit hat folgende Anwendungen:
- für Transfektionsverfahren
- für die Transfektion von Hek293T-Zellen
- für die Transfektion von H29D-Zellen
Geeignet für transiente wie für stabile Transfektionen von DNA in kultivierte Säugetierzellen. Bei den folgenden Zellen wurde eine erfolgreiche Transfektion mit der Calciumphosphat-Methode durchgeführt:
BAEC
Melanomzellen des Darms
CHO K1
COS-7
Fibroblasten (human embryonal, Neo Derm)
HEK293
Huh 7
IMR-90
LLC (Lewis-Lungenkarzinom)
NIH3T3
PC-12
PCI-13
SH-Sy5Y
SK-Hep-1
T47D
BAEC
Melanomzellen des Darms
CHO K1
COS-7
Fibroblasten (human embryonal, Neo Derm)
HEK293
Huh 7
IMR-90
LLC (Lewis-Lungenkarzinom)
NIH3T3
PC-12
PCI-13
SH-Sy5Y
SK-Hep-1
T47D
Biochem/physiol Actions
Bei diesem Verfahren wird eine HEPES-gepufferte Kochsalzlösung, die Natriumphosphat enthält, langsam mit einer CaCl2-Lösung gemischt, in der sich die DNA befindet. Es bildet sich ein DNA-Calciumphosphat-Co-Präzipitat, das an der Zelloberfläche anhaftet und von der Zelle aufgenommen wird, mutmaßlich mittels Endozytose.
Features and Benefits
- Geeignet für transiente wie für stabile Transfektionen
- Reproduzierbar für eine breite Palette von Zellarten
- Häufig referenziert
- Kostengünstig
Other Notes
Das Protokoll wurde entwickelt für die transiente Transfektion von CHO-Zellen unter Verwendung eines pSV40-CAT-Plasmids als Reportergen.
Lieferumfang des Calciumphosphat-Transfektionskits:
5 ml 2,5 M CaCl2 (C2052)
25 ml 2x HEPES-gepufferte Kochsalzlösung (H1012)
25 ml Wasser in Molekularbiologie-Qualität (W4502)
5 ml 2,5 M CaCl2 (C2052)
25 ml 2x HEPES-gepufferte Kochsalzlösung (H1012)
25 ml Wasser in Molekularbiologie-Qualität (W4502)
1 of 1
Dieser Artikel | ||
|---|---|---|
| description Most cost effective transfection reagent kit for transient and stable transfection of DNA into mammalian cells | description Lipid formulation for nucleic acid transfections in neuronal and glial cells | description powder, 4 μm, ≥80 m2g |
| technique(s) transfection: suitable | technique(s) transfection: suitable | technique(s) - |
| grade Molecular Biology | grade Molecular Biology | grade - |
| form solution | form dried film | form powder |
| Quality Level 200 | Quality Level 200 | Quality Level 100 |
| storage temp. −20°C | storage temp. 2-8°C | storage temp. - |
| shipped in dry ice | shipped in - | shipped in - |
Signalwort
Warning
Gefahrencodes
Sicherheitshinweise
Hazard Classifications
Eye Irrit. 2
Lagerklasse
12 - Non Combustible Liquids
Flammpunkt (°F)
Not applicable
Flammpunkt (°C)
Not applicable
WGK
WGK 1
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Is low cell passage number an important consideration for transfection?
1 answer-
Helpful?
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Is optimizing the transfection protocol important?
1 answer-
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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What quality does the DNA need to be in order to use it for transfection?
1 answer-
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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Can antibiotics be present in the medium during transfection?
1 answer-
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.
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How can I determine the efficiency of my transfection?
1 answer-
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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What is the Department of Transportation shipping information for this product?
1 answer-
Transportation information can be found in Section 14 of the product's (M)SDS.To access the shipping information for this material, use the link on the product detail page for the product.
Helpful?
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Is the size of the plasmid an important consideration for transfection?
1 answer-
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.
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What is transfection efficiency?
1 answer-
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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What is the difference between stable and transient transfection?
1 answer-
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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How can I increase the efficiency of my transfection?
1 answer-
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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