Plasmids used in this study are listed in Table S1. Oligonucleotides purchased from Integrated DNA Technologies are listed in Table S2. PCRs were carried out using AccuPrime Pfx or Taq (Thermo Fisher), purified using the NucleoSpin gel and PCR cleanup kit (TaKaRa, San Jose, CA), and cloned into plasmids using the In-Fusion HD cloning system (TaKaRa). Cloning reaction products were transformed into E. coli Stellar cells (TaKaRa) or PIR2 cells (Invitrogen) for plasmids with the R6K origin of replication. Oligonucleotides encoding targeting sgRNA were cloned into plasmid backbones with the NEBridge Golden Gate assembly kit (New England Biolabs [NEB]) as previously described (28 (link)).
Pir2 cells
Manufactured by Thermo Fisher Scientific
The PIR2 cells are a type of cell line used in various laboratory applications. They serve as a cellular model for research purposes, but a detailed description of their core function cannot be provided while maintaining an unbiased and factual approach without extrapolation on intended use.
Automatically generated - may contain errors
Lab products found in correlation
E coli stellar cells, by Takara Bio (2 mentions)
In fusion hd cloning system, by Takara Bio (2 mentions)
Accuprime pfx, by Thermo Fisher Scientific (2 mentions)
Nucleospin gel and pcr clean up kit, by Takara Bio (1 mentions)
Nebridge golden gate assembly kit, by New England Biolabs (1 mentions)
Oligonucleotide, by Integrated DNA Technologies (1 mentions)
2 protocols using pir2 cells
1
Molecular Cloning and Plasmid Construction
2
Plasmid Construction and Cloning Techniques
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Plasmids used in this study are listed in Table S1 . Oligonucleotides were purchased from Integrated DNA Technologies and are listed in Table S2 . PCRs were carried out using AccuPrime Pfx or Taq (Invitrogen). PCRs purified using the Nucleospin gel and PCR clean‐up kit were cloned into plasmid backbones digested with restriction enzymes (NEB) using the In‐Fusion HD cloning system (Takara). In‐Fusion reactions were transformed into E. coli Stellar cells (Takara) for non‐R6K origin of replication‐based vectors or PIR2 cells (Invitrogen) for R6K origin of replication plasmids. The sgRNA dsDNA fragments were cloned into plasmid backbones using the NEBridge Golden Gate Assembly kit (BsaI‐HF®v2) Construction of all the plasmids used in this study is detailed in File S1.
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