PCR was performed using Q5 High-Fidelity DNA Polymerase (New England Biolabs). All BE constructs were assembled using NEBuilder HiFi DNA Assembly (New England Biolabs). pCMV_ABEmax, ABE8e, pBT280, and SaKKH-ABEmax were a gift from David Liu (Addgene plasmid no. 112095, 138489, 122610, and 119815). pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT) was a gift from Akihiko Kondo (Addgene plasmid no. 79620). Plasmids expressing sgRNAs were cloned using T4 DNA Ligase (New England Biolabs). Lenti_gRNA-Puro was a gift from Hyongbum Kim (Addgene plasmid no. 84752). LentiGuide-Puro was a gift from Feng Zhang (Addgene plasmid no. 52963). sgRNA sequences used are listed in Table S1 .
Pcdna3.1 pcmv ncas pmcda1 ugi ph1 grna hprt
Manufactured by Addgene
PcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA(HPRT) is a plasmid vector used in molecular biology research. It contains a cytomegalovirus (CMV) promoter, a Cas9 nuclease, and a guide RNA (gRNA) sequence targeting the HPRT gene. The vector also includes a uracil glycosylase inhibitor (UGI) to enhance cytidine deaminase activity. The specific functions and intended uses of this vector should be evaluated by the researcher.
Automatically generated - may contain errors
Lab products found in correlation
Nebuilder hifi dna assembly, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Megaclear transcription clean up kit, by Thermo Fisher Scientific (1 mentions)
Pcmv be3, by Addgene (1 mentions)
Mmessage mmachine t7 ultra transcription kit, by Thermo Fisher Scientific (1 mentions)
Megashortscript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Herculase 2 fusion dna polymerase, by Agilent Technologies (1 mentions)
2 protocols using pcdna3.1 pcmv ncas pmcda1 ugi ph1 grna hprt
1
Genome Editing Toolkit Protocols
2
CRISPR-Based Gene Editing Protocols
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pCMV-BE3 (Addgene plasmid #73021), pBK-VQR-BE3 (Addgene plasmid #85171), and pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA (Addgene plasmid #79620) were obtained from Addgene. pCMV-BE3 and pBK-VQR-BE3 were gifts from David Liu. pcDNA3.1_pCMV-nCas-PmCDA1-ugi pH1-gRNA (HPRT) (Addgene plasmid #79620) was a gift from Akihiko Kondo. The mRNA template for BE, VQR-BE, and Target-AID were prepared by PCR using Herculase II Fusion DNA Polymerase (Agilent Technologies, #600675) with primers as listed in Supplementary Table 4 . BE, VQR-BE, and Target-AID mRNAs were synthesized using the mMESSAGE mMACHINE T7 Ultra Transcription Kit (Thermo Fisher Scientific, AM1345). Templates for in vitro transcription of sgRNAs against each of the Psen1 mutations and the App gene were prepared by PCR using Herculase II Fusion DNA Polymerase with primers as listed in Supplementary Table 5 . The sgRNAs were synthesized using the MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific, AM1354). Transcribed mRNAs and sgRNAs were purified using the MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific, AM1908).
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