What is the P3000 reagent?
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. For a more detailed description, check molecular mechanism of Lipofectamine 3000.
What is the role of P3000 reagent?
Lipofectamine 3000, when added to the mixture coats p3000 lipid droplets and confers a positive charge to the outside of the liposhperes. This positive charge allows endocytosis of the lipid droplets into the negatively-charged cell membrane.
What does Lipofectamine 3000 do?
Lipofectamine 3000 reagent leverages our most advanced lipid nanoparticle technology to enable superior transfection performance and reproducible results. It delivers exceptional transfection efficiency into the widest range of difficult-to-transfect and common cell types (Figure 1) with improved cell viability.
What is lipofectamine transfection?
Lipofectamine or Lipofectamine 2000 is a common transfection reagent, produced and sold by Invitrogen, used in molecular and cellular biology. It is used to increase the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection.
Is Lipofectamine a cationic?
Immunobiology of Dendritic Cells Part B Lipid-mediated transfection relies on cationic lipids, such as lipofectamine, which facilitate DNA and mRNA delivery into cells. The cationic lipids form a unilamellar liposomal structure with a positive surface charge and fuse with nucleic acids to form a transfection complex.
Can Lipofectamine be used in vivo?
As a prelude to direct in vivo gene transfer studies, we have compared the ability of two commercially available cationic lipids (Lipofectamine and DOSPER) to transfect C2C12 cells, a mouse muscle cell line in vitro. In addition, formu- lations suitable for in vivo injection were determined.
How do you mix lipofectamine?
Mix Lipofectamine 2000 gently before use, then dilute the appropriate amount in 250 μl of Opti-MEM I Medium (or other medium without serum). Mix gently and incubate for 5 minutes at room temperature. After 5 minutes incubation, combine the diluted DNA with the diluted Lipofectamine 2000 (total volume is 500 μl).
Is lipofectamine toxic to cells?
Although Lipofectin showed the lowest toxicity to HepG2 cells (89.54% viability), it displayed the lowest transfection efficacy too (8.29%). Generally, HepG2 cells displayed resistance to the toxicity of majority of the reagents tested, with Lipofectamine 3000 (70.59 % viability), being the most toxic reagent.
Does Lipofectamine expire?
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 does Lipofectamine increase transfection efficiency?
You do not even need to change the media after transfection. I use 2.5ul of Lipofectamine 3000 for each 1ug of plasmid each in 50ul optiMEM incubate for 20 min at room temp, then add to 1^6 of HEK293cells that plated in DMEM supplemented with 10% FCS, 48 hours later collect media. I got 90% efficiency.
When to perform a Lipofectamine 3000 transfection?
Transfection protocol On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24-well plate using Invitrogen ™ Lipofectamine 3000 Transfection Reagent: Step Tube Complexation components Amount per well (24-well) 1 Tube 1 Opti-MEM™ I medium 25 µL
Which is better Lipofectamine 3000 or FuGene HD?
Lipofectamine 3000 reagent outperforms Invitrogen Lipofectamine 2000 and FuGENE HD reagents. Each reagent was used to transfect HEK 293, HeLa, LNCaP, HepG2, and A549 cell lines in a 96-well format, and GFP expression was analyzed 48 hours posttransfection. Lipofectamine 3000 reagent provided higher GFP transfection efficiency than Lipofectamine
How much Lipofectamine is needed for genome editing?
A low toxicity lipid dose (0.75 µL for 24-well plates) is suggested for applications requiring minimal disruption of the cells. Lipofectamine® 3000 reagent is suitable for novel genome editing applications.