Preparing plasmids for nucleofection of hPSCs
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Published: 2022-09-07 DOI: 10.17504/protocols.io.b4pdqvi6
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Abstract
This protocol describes the standard procedure for the preparation of plasmids 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 plasmids On ice
2.
In each nucleofection, use 1 µg total of plasmid
3.
For prime editing PE2 strategy , use:
500 ng pCMV-PE2
500 ng pU6-pegRNA
4.
For prime editing PE3 strategy , use:
500 ng pCMV-PE2
330 ng pU6-pegRNA
170 ng pBPK1520-ngRNA
5.
Pipet the proper amount of each plasmid into a microcentrifuge tube. If the volume is too small, dilute in autoclaved water first.
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