How does PEI work in transfection?

How does PEI work in transfection?

DNA can be introduced into a host cell by transfection with polyethylenimine (PEI), a stable cationic polymer (Boussif et al., 1995). PEI condenses DNA into positively charged particles that bind to anionic cell surfaces. Our laboratory uses PEI over other cell transfection reagents because of its low cost.

How do you cook PEI for transfection?

Preparation of PEI Stocks

1. Dissolve 100 mg in 100 mL sterile ddH2O.
2. Stir while slowly adding HCl to pH 7.0.
3. Mix for 10 minutes and then recheck pH.
4. Filter sterilize through 0.22 um filter.
5. Aliquot 500 uL to 1000 uL and store in -80C.
6. Thawed solution can be stored at 4C for up to 2 months, label a tube when thawed.

How does calcium phosphate transfection work?

The calcium phosphate transfection method for introducing DNA into mammalian cells is based on forming a calcium phosphate-DNA precipitate. Calcium phosphate facilitates the binding of the DNA to the cell surface. DNA then enters the cell by endocytosis.

How long is PEI transfection?

Gently add the diluted PEI to the diluted DNA. Add the diluted PEI dropwise while gently flicking the diluted DNA tube. Incubate the mixture 15-20 min at room temperature. Carefully transfer the transfection mix to the Lenti-X 293T packaging cells.

What is the pH of calcium phosphate?

The results show that although calcium phosphate as such can reduce acidity, their buffering range lies at a pH of about 4, i.e. outside the physiological range. This is not related to the rate of dissolution. Carbonated calcium phosphates as well as calcium carbonate (calcite) alone are able to keep the pH around 7.4.

What is the use of PEI based transfection?

PEI-based transfection is widely used for transient transfection of plasmid DNA. The efficiency of PEI-based transfection is affected by numerous factors, including the way the PEI/DNA complex is prepared, the ratio of PEI to DNA, the concentration of DNA, the storage conditions of PEI solutions, and more.

How are GFP positive cells transfected with Pei?

Once a batch of PEI is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 days after transfection to determine what ratio gives the highest percentage of GFP positive cells. Gently add the diluted PEI to the diluted DNA. Add the diluted PEI dropwise while gently flicking the diluted DNA tube.

How is Pei used in HEK293 cell transfection?

HEK293 cell transfection procedure: For transfection, add the expression vector plasmid DNA to the cells at a final concentration of 3 µg/ml and PEI to a final concentration of 9 µg/ml of transfection volume. (DNA concentration may be varied from 1-6 µg/ml to optimize expression, while PEI is maintained at 9 µg/ml).

Which is the best protocol for Lipofectamine reagent?

Our Lipofectamine Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. This is often the best place to start, especially in a new cell line. If you find this doesn’t work for your specific cell type, then you can you look to our cell specific protocols for further optimization.