Green Lab Nanoparticle For 6 Well Cardiomyocyte Transfection v2
Edwin Yoo
Published: 2023-03-07 DOI: 10.17504/protocols.io.ewov1ojd2lr2/v2
Abstract
nanoparticle transfection
Steps
Nanoparticle Synthesis
1.
Defining Terms
- DNA refers to nucleic acid cargo encoding genes of interest, generally stored at a concentration of 1 ug/ul.
- Polymer refers to the Poly(beta-amino ester) (PBAE) that makes up the backbone of the nanoparticle.
- Nanoparticle refers to the final complexed polymer + DNA that is ready for transfection.
2.
DNA Dilution
For 3 technical replicate wells of one condition in 6-well plate format, mix the following in microcentrifuge tube.
- 60 ul DNA at 1ug/ul
- 440 ul sodium acetate
- Total volume = 500 ul for 3 technical replicate wells per one condition
3.
Polymer Dilution (7.2 mg.ml polymer concentration in NaAc)
For 3 technical replicate wells of one condition in 6-well plate format, mix the following in microcentrifuge tube.
- 40 ul polymer (e.g. 4-5-6)
- 460 ul sodium acetate
- Total volume = 500 ul for 3 technical replicate wells per one condition
4.
Nanoparticle (DNA+polymer) Synthesis
Combine diluted DNA with diluted polymer
- Add 500 ul of diluted DNA to 500 ul diluted polymer (can freeze here at -80 at your own risk)
- Add 5000 ul of serum containing media to 1000 ul NP's for final volume master mix of 6000 ul per condition
- Incubate for 10 minutes
5.
Transfection
- Change media from each well of 6 well plate with 2000 ul nanoparticle master mix (media + nanoparticles)
- Incubate for 24 hours or desired time
- Change media after desired transfection time.
