What does PEI do in transfection?
4 Incubate the mixture at room temperature for 30 min. 2.2. 5 Add the PEI-DNA mixture to the suspension cells from Step 2.1. 8.
How do you use transfection on PEI?
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.
How do you transfect cell protocols?
In a standard transfection protocol, the cells are plated on day 1, transfected on day 2 and assayed on day 3 or 4. In a reverse transfection protocol, cells are added directly to a plate containing the transfection reagent/DNA mix and assayed on day 2 or 3.
What are the three main types of chemical transfection reagents?
Summary of the commonly used chemical transfection reagents. Transfection reagents can be generally divided into liposomal or high-lipid based and non-liposomal based reagents. Non-liposomal reagents can be mixed protein-lipid reagents, non-liposomal lipids or non-protein non-lipid reagents like dendrimer.
How does PEI work?
PEI condenses DNA into positively charged particles, which bind to anionic cell surface residues and are brought into the cell via endocytosis. Once inside the cell, protonation of the amines results in an influx of counter-ions and a lowering of the osmotic potential.
What does PEI Pro do?
Summary. PEIpro® product range (PEIpro®, PEIpro®-HQ and PEIpro®-GMP) is the transfection method of choice to achieve reliable viral vector production and high infectious titer yields, with direct scalability and seamless transition from process development up to large-scale clinical-grade manufacturing.
How do you make a PEI transfection reagent?
Preparation
- Dissolve 100 mg in 100 mL sterile ddH2O.
- Stir while slowly adding HCl to pH 7.0.
- Mix for 10 minutes and then recheck pH.
- Filter sterilize through 0.22 um filter.
- Aliquot 500 uL to 1000 uL and store in -80C.
- Thawed solution can be stored at 4C for up to 2 months, label a tube when thawed.
How do you make a PEI solution for transfection?
Preparation of PEI Stocks
- Dissolve 100 mg in 100 mL sterile ddH2O.
- Stir while slowly adding HCl to pH 7.0.
- Mix for 10 minutes and then recheck pH.
- Filter sterilize through 0.22 um filter.
- Aliquot 500 uL to 1000 uL and store in -80C.
- Thawed solution can be stored at 4C for up to 2 months, label a tube when thawed.
What is PEI pro?
PEIpro® is a ready to use 1 mg/ml optimised linear polyethylenimine (PEI) reagent for the production of virus & recombinant proteins by Transient Gene Expression (TGE) in suspension-adapted mammalian cell lines cultivated in shaker flasks, platform shakers or stirred tank bioreactors.
What is PEI coating?
PEI (Polyetherimide), also known as Ultem, is a reusable “relatively maintenance free” build surface for both ABS (with a Heated Bed) and PLA (hot or cold) requiring no additional adhesives such as glue or tape. PEI is appealing due to its “no-surface-prep” conditions, which make it convenient to work with.
How to test for the transfection of Pei?
Count the cells using a hemacytometer. For each transfection condition to be tested, aliquot 3 × 10 5 cells into a sterile centrifuge tube. Spin the cells at 200 × g at room temperature for 5 min. Aspirate supernatant. Add medium to the cell pellet to a final concentration of 3 × 10 5 cells ml −1.
Why is Pei used to transfect suspension cells?
Our laboratory uses PEI over other cell transfection reagents because of its low cost. This protocol is appropriate for two suspension cell lines, CHO-S and HEK 293 GnTi-. Many cell lines can be transfected successfully with PEI but in our experience these two cell lines express the highest level of protein compared to other cells. 2. EQUIPMENT
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.
What is the transfection protocol for polyethylenimine?
Polyethylenimine (PEI) transfection protocol 1 Linear Polyethylenimine (L-PEI) MW 25.000 2 1 M NaOH 3 PBS 4 Trypsin 5 Gibco CO2 independant medium + 10% FBS and L-Glutamine 6 Plasmid DNA containing gene of interest