PEI Transfection Reagent (HY-K2014, MCE)
Hello Yan Yan Zhao,
1. Before plasmid transfection, do 293T cells need to be cultured in serum-free medium for 1 hour before adding the transfection reagent-nucleic acid complex?
No. In PEI-mediated transfection of 293T cells, a period of 1 hour of serum-free incubation is not required before adding the transfection complex. The plasmid DNA and PEI are diluted separately in a small volume of serum-free medium (such as Opti-MEM). This is because serum proteins can interfere with the formation of the DNA-PEI complex, but once the complexes have formed, serum does not pose a problem for cellular uptake.
PEI is then added to the DNA solution, mixed, and incubated for a short time (typically 10–20 minutes) at room temperature to allow the complexes to form.
2. If I want to transfect multiple plasmids, should the two plasmids be added to the same tube of transfection reagent, or should they be added separately into two tubes?
You should combine the two plasmids in a single tube before adding the transfection reagent. This method ensures that the transfection complexes contain a mix of both plasmids, maximizing the chances that a single cell will take up both.
On the other hand, if they are added separately into two tubes it may result in decorrelated delivery. Some cells will take up only plasmid 1, others only plasmid 2, and some will take up both. This creates a range of different plasmid ratios inside the cell.
So, adding the two plasmids to the same tube of transfection reagent will ensure that most transfected cells receive and express both plasmids simultaneously. Please note that this method will maximize the chance of both being delivered, but it is not a guarantee of 100% co-delivery.
Best,
Hi Yan Yan Zhao
- Serum can affect the formation of the transfection reagent–DNA complex. For 293T cells, you can switch to serum-free medium for about 1 hour before adding the transfection reagent–nucleic acid complex. After transfection, replace it with complete culture medium appropriate for your cells.
- In general, when co-transfecting plasmids, the two plasmids are usually mixed in the same tube first, and then combined with the transfection reagent before being added to the cells. However, the ratio of the two plasmids may need to be optimized experimentally to achieve good transfection efficiency and quality.
The answer to this question is provided by MedChemExpress Technical Support.
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