Tips and tricks

What is the best transfection reagent for HEK293?

What is the best transfection reagent for HEK293?

Conclusion: Lipofectamine 3000 is the best choice for transfection of CHO-K1 and HEK293 with pCDH while Turbofect is preferably used in transfecting these cell lines with pEGFP-N1 (Tab.

What are the two chemical reagents that can be used for transfection?

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 do you transfect an A549 cell?

Transfecting A549 Cells Plate 4 x 104cells per well in 0.5 ml of complete growth medium. Cell density should be 50~80% confluent on the day of transfection. For each well of cells to be transfected, dilute 0.5 μg of DNA into 100 μl of Opti-MEM® I Reduced Serum Medium without serum.

Does EDTA interfere with transfection?

In this study, we evaluated the EDTA as a new transfection method and confirmed both improved transfection efficiency and stable cell viability on hESCs. These results demonstrate that usage of EDTA is safe substitute for transfection than that of enzymatic methods.

What is PEI reagent?

Polyethylenimine (PEI) is a simple, inexpensive and effective reagent for condensing and linking plasmid DNA to adenovirus for gene delivery. Gene Ther.

How can I increase my transfection efficiency?

Improving the Success of Your Transfection

  1. Transfect healthy, actively dividing cells at a consistent cell density.
  2. Transfect using high-quality DNA.
  3. Optimize the amount of DNA used to transfect cells.
  4. Optimize the transfection reagent:DNA ratio.
  5. Optimize cell number per well when transfected.

Are A549 cells easy to transfect?

Superior Tranfection Efficiency and Maximum Cell Viability In a head-to-head comparison study, DNA-In A549 Reagent consistently outperformed Lipofectamine® 2000 and Lipofectamine® 3000 transfection reagents. Above, A549 cells were plated at 70% confluency and were transfected 24 hours after plating.

What is A549 cells?

A549 cells are lung carcinoma epithelial cells that constitute a cell line. A549 is one of the commonly used cell lines for a wide range of research applications.

How do you increase transfection efficiency of Lipofectamine 3000?

  1. The day before transfection, Plate 4×105 cells per well in 0.5 mL of complete growth medium.
  2. For each well of cells to be transfected, dilute 1 μg of DNA in 50 μL of Opti-MEM , and 2.5 μL of P3000 in 50 μL of Opti-MEM in separated tube.
  3. Mix gently and incubate 20 minutes at room temperature.

What does P3000 reagent do?

P3000 reagent: This reagent will be added to the diluted DNA solution, and its function is to help nucleic acids entering both the cell and the nucleus.

What is the advantage of using HEK293T cells in this assay?

Advantages of HEK293 Reproducibility of results: A key advantage of this cell line is that results are generally consistent and highly reproducible. Gene Expression: HEK cells can be used for both transient and stable expression of desired genes.

Why is HEK293 used for transfection?

Advantages of HEK293 cells Firstly, they are very easy to grow and to maintain, with high reproducibility, which makes them preferable over other less-robust and slow-growing cell lines. Furthermore, they are very efficient at protein production and accessible for transfection.

Which PEI is used for transfection?

polycation polyethylenimine
The polycation polyethylenimine (PEI) is one of the most utilized reagents for small- to large-scale transfections as it is simple to use and, when combined with optimized expression vectors and cell lines, provides high transfection efficiency and titers.

Why is PEI used in transfection?

PEI condenses DNA into positively charged particles that bind to anionic cell surfaces. Consequently, the DNA:PEI complex is endocytosed by the cells and the DNA released into the cytoplasm (Sonawane et al., 2003). Our laboratory uses PEI over other cell transfection reagents because of its low cost.

How long do A549 cells take to adhere?

24-40 hours
A549 Cell Subculture Protocol A549 cells are cultured in complete media consisting of Dulbecco’s MEM modified with 10% FBS. The cells can be grown as adherent or in suspension in vitro. The A549 cell line grows easily and cell count doubling time is typically 24-40 hours.

Can A549 cells be grown in DMEM?

Growth Medium: The A549 cells are typically cultured using a base medium F12/K (Gibco/Invitrogen). 10% fetal bovine serum (FBS) is added to the base medium to make the complete growth medium. The cells can also be cultured in complete media consisting of Dulbecco’s MEM (DMEM) modified with 10% FBS.

Why do we use A549 cells?

The A549 cells have been used to model the alveolar Type II pulmonary epithelium. Studies have shown that this can be particularly useful in research for studying the metabolic processing of lung tissue and for identifying mechanisms of drug delivery to the tissue.

Is P3000 reagent necessary?

There is no need to use the P3000™ Reagent.

What is the difference between 293 and 293T cells?

The 293T cells are derived from 293 cells but stably express the SV40 large T antigen which can bind to SV40 enhancers of expression vectors to increase protein production. 293T are also neomycin resistant due to the presence of a neomycin resistance cassete together with the SV40 Large T .