How do you measure GFP transfection efficiency?
Using an appropriate promoter, GFP can be expressed in the cells by itself or attached to the protein of interest as a fusion protein. With a GFP reporter system, the transfection efficiency can easily be determined as the percentage of cells expressing GFP in the entire cell population.
What is a good transfection efficiency?
With cationic lipid-mediated transfection, generally 70–90% confluency for adherent cells or 5 × 105 to 2 × 106 cells/mL for suspension cells at the time of transfection provides good results.
How do you calculate transfection efficiency?
One general method for measuring transfection efficiency is to use a fluorescence microscope. The transfection efficiency is measured by counting the total number of observed cells and the number of cells that express fluorescence, and scoring these values.
What is GFP plasmid?
The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms. The plasmid contains several reporter genes, most notably the green fluorescent protein (GFP) and the ampicillin resistance gene. GFP was isolated from the jelly fish Aequorea victoria.
What is Lipofectamine transfection?
Lipofectamine or Lipofectamine 2000 is a common transfection reagent, produced and sold by Invitrogen, used in molecular and cellular biology. It is used to increase the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection.
Can Lipofectamine be used in vivo?
As a prelude to direct in vivo gene transfer studies, we have compared the ability of two commercially available cationic lipids (Lipofectamine and DOSPER) to transfect C2C12 cells, a mouse muscle cell line in vitro. In addition, formu- lations suitable for in vivo injection were determined.
Why is GFP used in transfection?
Transient transfection is often used to study gene expression or promoter func- tions in mammalian cells into which plasmid constructs have to be delivered. Perhaps the most important advantage is that the GFP tag allows the monitoring of gene expression within living cells.
How do you validate a transfection?
A common way to validate that a genetic material was successfully introduced into cells is to measure protein expression. This is typically performed by Western blot or immunostaining, however, TaqMan® Protein Assays offer a faster, more sensitive and more quantitative alternative to these techniques.
Why is my transfection efficiency low?
This is because cells that are actively dividing take up DNA more readily than stationary phase cells. If cells are allowed to become fully confluent, they begin to exhibit contact inhibition and cease actively dividing – this limits their ability to uptake nucleic acids and negatively impacts transfection efficiency.
Is Lipofectamine stable transfection?
Lipofectamine® 3000 reagent leverages our most advanced lipid nanoparticle technology to enable superior transfection performance and reproducible results. It delivers exceptional transfection efficiency into the widest range of difficult-to-transfect and common cell types with improved cell viability.
Can Lipofectamine 3000 enhance green fluorescent protein (EGFP)?
Enhanced green fluorescent protein (EGFP) was clearly seen in Eppendorf tubes from harvested cells using Lipofectamine 3000 without using a microscope and UV activation. Strong expression of EGFP was observed in HEK293 cells, mouse primary cortical neurons and human umbilical vein endothelial cells (HUVECs) using confocal microscopy.
What is the best reagent for GFP transfection?
Invitrogen product line such as Lipofectamine 2000 reagent and Lipofectamine 3000 reagent were used to transfect 17 cell lines with a GFP-expressing plasmid in a 24-well plate format, using 0.5 µg plasmid/well and the recommended protocols for each reagent. GFP expression was analyzed 48 hours posttransfection.
What is the best ratio of plasmid to Lipofectamine for transient lipofection?
We found that the most performing ratio of plasmid:lipofectamine was 10:50 for transient lipofection, whereas two pulses for 10 s at 960 microF of capacitance, 200 V of voltage were the most favorable electrical parameters for EP efficiency in the presence of 5 microL of phMGFP plasmid.
Which Lentivirus transfection reagent has the highest GFP expression?
It showed the highest GFP expression using lentivirus generated with Lipofectamine 3000 transfection reagent (A) as compared with using Lipofectamine 2000 (B) or FuGENE 6 (C) transfection reagents. EGFP is clearly seen without using a microscope or without an UV stimulation condition