How much DNA is needed to transfect?
Optimal amount of Universal Transfection Reagent used depends on cell type and is generally 1 – 3 µL per ug of plasmid DNA.
How confluent should cells be for transfection?
Confluency. As a general guideline, transfect cells at 40–80% confluency. Too few cells will cause the culture to grow poorly without cell-to-cell contact. Too many cells results in contact inhibition, making cells resistant to the uptake of foreign DNA.
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 do you optimize transfection efficiency?
Use any GFP plasmid DNA to optimize the condition to see transfection efficiency and toxicity. OptiMEM is the best media to use for transfection, but this is just used for complex formation between the reagent and the DNA.
What happens if cells become too confluent?
If you passage at low confluency, your cells will be selected for faster growing cells over time. On the other hand, with high confluency the gene expression due to contact inhibition will be changed .
Can I split cells after transfection?
I usually split the cells (1:40, or 1:80) 24 hours after transfection, then directly add the drugs. The dilution ratio depends on the transfection efficiency and cell doubling time. It’s better to use the ratio which can give single isolated colonies on the plates. Change the medium every 2 days.
How do you measure 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.
Does plasmid size affect transfection efficiency?
True. If your plasmid size is big, it would make hard sometimes for transfection.
How is transfection efficiency calculated?