How much DNA does it take to transfect a 10cm dish?
Total DNA amount used in calcium phosphate transfection is usually 10–50 μg in 450 μL sterile water and 50 μL of 2.5 M CaCl2 per 10-cm dish, but varies widely among plasmid preparations as well as with different cells and media.
How much DNA does it take to transfect a 6 well plate?
Typically, for experiments in 6-well plates, 150 000 to 250 000 cells are seeded per well 24 h prior to transfection. For other culture formats, refer to Table 1. For DNA/siRNA co-transfection experiments, we recommend using 2 µg DNA and 10 to 50 nM siRNA per well in a 6-well plate.
What is FuGENE 6 transfection reagent?
FuGENE® 6 Transfection Reagent(a) is a nonliposomal reagent that transfects DNA into a wide variety of cell lines with high efficiency and low toxicity. The protocol does not require removal of serum or culture medium and does not require washing or changing of medium after introducing the reagent/DNA complex.
What is PEI 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.
What is FuGENE transfection reagents and how does it work?
FuGENE® HD Transfection Reagent is a multi-component reagent that forms a complex with DNA, then transports the complex into animal or insect cells. It is suitable for use in media with or without serum, and for transient or stable transfection, as well as co-transfections of multiple DNA plasmids.
What is FuGENE transfection reagent?
Can I use expired Lipofectamine?
According to ThermoFisher, lipofectamine 3000 can be used until 2 years after the manufacturing date if stored properly (stored in a 4 degree fridge should be fine). The Optimem is sensitive to expiration. I would recommend using the new reagents for your experiment. Hope this helps!
How do you cook PEI 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.
How big is the plate for Universal transfection reagent?
Adherent Culture Tube A Tube A Tube B 12-well plate 1 1.5 100 4.5 6-well plate 2 2 150 6 3.5 cm dish 2 3 100 9 6 cm dish 3 5 250 15
When to change the media after a transfection?
A complete media change can be performed 5 – 24 hours after transfection for very sensitive cells. For most cells, we recommend the media be changed only at 48 hours post-transfection until protocol optimization requires this extra media change. Day 4: Change Medium and Check Transfection Efficiency
How big is a cell culture flask dish?
Useful information for various sizes of cell culture dishes and flasks Catalog No. Surface area (cm 2) Seeding density* Cells at confluency* 35mm 150460; 150318 8.8 0.3 x 10 6 1.2 x 10 6 60mm 150462; 150288 21.5 0.8 x 10 6 3.2 x 10 6 100mm 150464; 150350 56.7 2.2 x 10 6 8.8 x 10 6
