Transfection with PEI

实验概要

Use PEI (Linear 25 kDa Reagent) to transfect in HEK293T cells.

主要试剂

PEI is polyethyleimine, a 25 kDa linear from Polysciences Inc. Make up solution at 1μg/μl in sterile water, neutralize with HCl to pH 7.2 and filter sterilize using a 0.22μm filter. Aliquot and store at -80℃. A 5-10ml aliquot can be kept at 4℃ for up to 4 months.

实验步骤

6-well plate:per well
1. Seed about 6 *105 cells 24 hrs before transfection;

2. Next day, the cells should achieve 60% confluency. Change the medium before transfection. This is required since there is something that most cells secrete that will interfere with the reagent.(Not always necessary)

3. In an eppendorf tube, dilute 3μg total in 200μl of serum-free media(Simply DMEM or Opti-MEM). Mix well by pipetting up and down.

4. Add PEI to the diluted DNA and vortex for 10 seconds immediately. Optimize for cells by trying 1:1, 2:1, and 3:1 ratios of PEI:DNA.

5. Incubate for 15 minutes at room temperature.

6. Add DNA/PEI mix evenly over the 6-well dish. After adding all the samples, gently swirl plate for a few seconds to mix the components.

注意事项

Tips:

PEI is suitable for 293T、COS-7、U2OS1、H1299 transfection.

For 293T cells, the efficiency is over 80% at 1:1 ratio of PEI:DNA.

For 10cm plate, 1ml serum-free medium, 6~10μg DNA, and 1:1 PEI:DNA is recommended.