Preparing mRNA for nucleofection of hPSCs
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Published: 2022-09-07 DOI: 10.17504/protocols.io.b4peqvje
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Abstract
This protocol describes the standard procedure for preparing mRNA to be delivered into human pluripotent stem cells (hPSCs) using nucleofection.
General notes
- Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
Steps
1.
Thaw mRNA and synthetic pegRNA/ngRNA On ice
2.
In each nucleofection, use 4 µg total of mRNA
3.
For prime editing PE2 strategy , use:
4 µg, IVT PE2 mRNA
1.5 µl, 100 µM Synthetic pegRNA
4.
For prime editing PE3 strategy , use:
4 µg, IVT PE2 mRNA
1 µl, 100 µM Synthetic pegRNA
0.5 µl, 100 µM, Synthetic ngRNA
5.
Pipet the proper amount of each component into a microcentrifuge tube.
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