How long can you incubate the plasmid DNA with Lipofectamine reagent?

Incubate the cells at 37°C in a CO2 incubator for 24-96 hours until you are ready to assay for gene knockdown. Medium may be changed after 4-6 hours.

How long does Lipofectamine take to work?

Depending on the construct used, transiently expressed transgene can generally be detected for 1 to 7 days, but transiently transfected cells are typically harvested 24 to 96 hours post-transfection.

What is the difference between lipofectamine 2000 and 3000?

Lipofectamine 3000 reagent outperforms Invitrogen Lipofectamine 2000 and FuGENE HD reagents. Lipofectamine 3000 reagent provided higher GFP transfection efficiency than Lipofectamine 2000 and FuGENE HD reagents for all five cell lines.

How long after transfection is GFP?

GFP expression was noticeable in cells within 4 h of transfection. In nine separate transfections, approximately 20% of the transfected cells expressed GFP with a mean fluorescence 40-50x that of control cells (15 fluorescent units [FU] vs. 0.3 FU) during the first five days after transfection.

Can you 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.

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.

How do you increase lipofectamine 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.

What does p3000 reagent do?

How long after transfection can I harvest cells?

Cells are typically harvested 24–72 hours after transfection for studies designed to analyze transient expression of transfected genes.

When to use Lipofectamine 2000 as a reagent?

Researchers use Lipofectamine® 2000 Reagent for siRNA- and shRNA-based gene knockdown experiments, as well as for gene expression studies. Lipofectamine® 2000 Transfection Reagent works effectively with all common cell lines as well as with many challenging ones, and can be used in media with or without serum.

What is the transfection protocol for Lipofectamine LTX?

Lipofectamine® LTX & PLUS™ Reagent Protocol Outline A. Plate cells so they will be 70–90% confluent at the time of transfection. B. Prepare plasmid DNA-lipid complexes. C. Add DNA-lipid complexes to cells. Lipofectamine® LTX DNA Transfection Reagent Protocol See page 2 to view a typical plasmid transfection procedure. Transfection Amounts

How to transfect plasmid DNA with Lipofectamine 2000?

Transfect plasmid DNA and siRNA at the same time using Lipofectamine® 2000 Reagent by adding 30 pmol (~0.6 μg) of siRNA per 1 μg of DNA. mRNA can be transfected in a 24-well plate using Lipofectamine® 2000 Reagent by adding 0.5–1 μg of mRNA per well. Store at 4°C (do not freeze).

What kind of cells can Lipofectamine be used with?

When used in combination with PLUS™ Reagent, Lipofectamine® Transfection Reagent has been shown to work well with cells such as BHK-21, NIH 3T3, COS-1, fibroblasts, keratinocytes, HT-29, MRC-5, and SK-BR3. With such a legacy comes a commitment to providing the best reagents for all scientists and labs, regardless of their funding capabilities.