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Bsmbi digestedacceptor vector

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BsmBI-digested acceptor vector is a plasmid designed for molecular cloning. It contains a BsmBI restriction site, which can be used to insert DNA fragments into the vector through a cut-and-paste process. The vector provides a framework for the insertion and propagation of foreign DNA sequences in host organisms, such as bacteria, for various research applications.

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Lab products found in correlation

Q5 hot start high fidelity 2x master mix, by New England Biolabs (2 mentions) Cy5 labeled dna oligonucleotides, by Integrated DNA Technologies (2 mentions) Dcas9 protein, by Integrated DNA Technologies (2 mentions) Cas9 h840aprotein, by Integrated DNA Technologies (2 mentions) Usingplasmid plus midiprep kits, by Qiagen (2 mentions) Pureyield plasmid miniprep kit, by Promega (2 mentions) Phusion u green multiplex pcr master mix, by Thermo Fisher Scientific (2 mentions) Wild type c57bl 6 mice, by Charles River Laboratories (2 mentions) Q5 dna polymerase, by Thermo Fisher Scientific (1 mentions)

3 protocols using bsmbi digestedacceptor vector

1

Plasmid Construction for CRISPR Editing

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DNA amplification was conducted by PCR using Phusion U Green Multiplex
PCR Master Mix (ThermoFisher Scientific) or Q5 Hot Start High-Fidelity 2x Master
Mix (New England BioLabs) unless otherwise noted. DNA oligonucleotides,
including Cy5-labeled DNA oligonucleotides, dCas9 protein, and Cas9 H840A
protein were obtained from Integrated DNA Technologies. Yeast reporter plasmids
were derived from previously described plasmids39 and cloned by the Gibson assembly
method. All mammalian editor plasmids used in this work were assembled using the
USER cloning method as previously described40 . Plasmids expressing sgRNAs were constructed by
ligation of annealed oligonucleotides into BsmBI-digested
acceptor vector (Addgene plasmid #65777). Plasmids expressing pegRNAs were
constructed by Gibson assembly or Golden Gate assembly using a custom acceptor
plasmid (see Supplementary
Note 3). Sequences of sgRNA and pegRNA constructs used in this work
are listed in Supplementary
Tables 2 and 3. All vectors for mammalian cell experiments were purified using
Plasmid Plus Midiprep kits (Qiagen) or PureYield plasmid miniprep kits
(Promega), which include endotoxin removal steps. All experiments using live
animals were approved by the Broad Institute Institutional and Animal Care and
Use Committees. Wild-type C57BL/6 mice were obtained from Charles River
(#027).
Anzalone A.V., Randolph P.B., Davis J.R., Sousa A.A., Koblan L.W., Levy J.M., Chen P.J., Wilson C., Newby G.A., Raguram A, & Liu D.R. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576(7785), 149-157.
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2

SBDS Minigene Construction and Variants

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To create the SBDS minigene, the genomic region of the human SBDS gene (LRG_104; NM_016038.4) including the last 578 bp of intron 1, exon 2 (130 bp), and the first 442 bp of intron 2 was amplified from the genomic DNA of a healthy subject using high-fidelity Q5 DNA-Polymerase (ThermoFisher Scientific, Waltham, MA, USA) and primers SBDS IVS1 NdeI F and SBDS IVS2 NdeI R, and subsequently cloned into the pTB plasmid by exploiting the NdeI restriction site inserted within primers. The c.258+2T>C variant was introduced by site-directed mutagenesis. Expression vectors for the U1snRNA and U7snRNA variants were created as previously reported [6 (link)]. Plasmids expressing sgRNAs and pegRNAs were constructed by the ligation of annealed oligonucleotides into the BsmBI-digested acceptor vector (Addgene plasmid no. 65777) and BsaI-digested acceptor vector (Addgene plasmid no. 132777), respectively.
To create the pre-mRNA trans-splicing molecules (PTMs), the coding sequence of EGFP (without the first two nucleotides of the starting codon) was fused with the optimized 3′ portion of IVS1 of the HBB gene. The binding domain (BD), cloned in the correct or opposite orientation as the experimental control, was inserted between the HindIII and BamHI restriction sites.
All plasmids were validated by Sanger sequencing. All primer sequences are listed in Table S1.
Peretto L., Tonetto E., Maestri I., Bezzerri V., Valli R., Cipolli M., Pinotti M, & Balestra D. (2023). Counteracting the Common Shwachman–Diamond Syndrome-Causing SBDS c.258+2T>C Mutation by RNA Therapeutics and Base/Prime Editing. International Journal of Molecular Sciences, 24(4), 4024.
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3

Plasmid Construction for CRISPR Editing

Check if the same lab product or an alternative is used in the 5 most similar protocols
DNA amplification was conducted by PCR using Phusion U Green Multiplex
PCR Master Mix (ThermoFisher Scientific) or Q5 Hot Start High-Fidelity 2x Master
Mix (New England BioLabs) unless otherwise noted. DNA oligonucleotides,
including Cy5-labeled DNA oligonucleotides, dCas9 protein, and Cas9 H840A
protein were obtained from Integrated DNA Technologies. Yeast reporter plasmids
were derived from previously described plasmids39 and cloned by the Gibson assembly
method. All mammalian editor plasmids used in this work were assembled using the
USER cloning method as previously described40 . Plasmids expressing sgRNAs were constructed by
ligation of annealed oligonucleotides into BsmBI-digested
acceptor vector (Addgene plasmid #65777). Plasmids expressing pegRNAs were
constructed by Gibson assembly or Golden Gate assembly using a custom acceptor
plasmid (see Supplementary
Note 3). Sequences of sgRNA and pegRNA constructs used in this work
are listed in Supplementary
Tables 2 and 3. All vectors for mammalian cell experiments were purified using
Plasmid Plus Midiprep kits (Qiagen) or PureYield plasmid miniprep kits
(Promega), which include endotoxin removal steps. All experiments using live
animals were approved by the Broad Institute Institutional and Animal Care and
Use Committees. Wild-type C57BL/6 mice were obtained from Charles River
(#027).
Anzalone A.V., Randolph P.B., Davis J.R., Sousa A.A., Koblan L.W., Levy J.M., Chen P.J., Wilson C., Newby G.A., Raguram A, & Liu D.R. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576(7785), 149-157.
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