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.

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

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

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

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.