Mar 07, 2023

Public workspaceGreen Lab Nanoparticle For 6 Well Cardiomyocyte Transfection v2 V.2

DOI
dx.doi.org/10.17504/protocols.io.ewov1ojd2lr2/v2
  • 1johns hopkins
Edwin Yoo
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.ewov1ojd2lr2/v2
Protocol Citation: Edwin Yoo 2023. Green Lab Nanoparticle For 6 Well Cardiomyocyte Transfection v2. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1ojd2lr2/v2Version created by Edwin Yoo
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 07, 2023
Last Modified: March 07, 2023
Protocol Integer ID: 78272
Abstract
nanoparticle transfection
Materials
Sodium acetate 25mM pH5
Polymer stock (100 ug/ul)
Desired media
Nanoparticle Synthesis
Nanoparticle Synthesis
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.