pSpCas9(BB)02A-Puro (PX459) V2.0 was a gift from Dr Feng Zhang (Addgene plasmid #62988) and ligated with annealed oligos containing restriction sites and sgRNA target sequence to generate the PX459-sgRNA F73. pMK286 (mAID-NeoR) was a gift from Dr Masato Kanemaki (Addgene plasmid #72824). pAcGFP-C1-RPA123-P2A was a gift from Dr Claudia Lukas (13 (link)). Homology sequences to the C-terminal end of TIM was obtained through gene synthesis and ligated to pUC57 (Gene Universal), which was used to generate the pUC57-mAID-NeoR donor template using Gibson assembly. Four mutations in the target sequence were made during gene synthesis and an additional PAM site mutation was generated through site-directed mutagenesis (SDM). pcDNA3-Flag-MRE11 was synthesized from Genscript (SC1200/M). Primers containing overlapping homology sequences or mutations were used for Gibson Assembly and SDM, respectively. The ligated products were transformed into DH5α competent cells, with the exception of the px459-sgRNA F73 which was transformed into NEB Stable (C3040H; New England BioLabs) competent cells. Individual colonies were inoculated in Luria-Bertani (LB) media for DNA extraction using DNA miniprep or midiprep kits (Promega). All mutations and plasmid sequences were verified by Sanger DNA sequencing (Stony Brook University Genomic Facility).
Neb stable
Manufactured by New England Biolabs
Sourced in United States
NEB Stable is a laboratory equipment product designed to preserve and protect biological samples during storage and transportation. It provides a stable environment for maintaining the integrity of various biomolecules, such as proteins, enzymes, and nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Neb 5 alpha, by New England Biolabs (3 mentions)
Hifi dna assembly, by New England Biolabs (2 mentions)
Q5 dna polymerase, by New England Biolabs (2 mentions)
Fastgene plasmid mini kit, by Nippon Genetics (2 mentions)
Lr clonase 2, by Thermo Fisher Scientific (2 mentions)
Pentr221, by Thermo Fisher Scientific (2 mentions)
Pmkate2 h2b, by Evrogen (2 mentions)
Bp clonase 2, by Thermo Fisher Scientific (2 mentions)
Pdonr221, by Thermo Fisher Scientific (2 mentions)
Stbl3, by Thermo Fisher Scientific (2 mentions)
Midiprep kit, by Promega (1 mentions)
Dna miniprep, by Promega (1 mentions)
Platinum gate talen kit, by Addgene (1 mentions)
In fusion snap assembly master mix, by Takara Bio (1 mentions)
Dpni, by New England Biolabs (1 mentions)
Phusion dna polymerase, by New England Biolabs (1 mentions)
Q5 polymerase, by New England Biolabs (1 mentions)
Nebuilder hifi, by New England Biolabs (1 mentions)
12 protocols using neb stable
1
CRISPR-Cas9 Mediated Gene Knockout
2
Cloning Vector Preparation and Transformation
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Prepared vector (cut, dephosphorylated, and PEG purified) was diluted to a working concentration of ~20–100 ng/μL if needed. Phosphorylated oligos were diluted (from the heat-inactivated PNK reaction) to a 1 ng/μL working concentration. Ligations were performed using the LigateIT rapid ligase kit (78400, Affymetrix) with 100 ng vector DNA and an 8:1 insert:vector molar ratio. A vector-only ligation was also prepared to control for incompletely digested and/or re-ligated vector derived colonies. Next, 2 μL of the ligation reactions were transformed into Stbl3 (Life Technologies) or NEB-Stable (NEB) chemically competent E. coli. Competent cells were incubated on ice for 30 min with 4 μL of ligation DNA, then heat shocked at 42 °C for 40 s and returned to ice for 1 min. Then, 1 mL of LB media was then added to the cells and they were allowed to recover at 37 °C for 30 min, after which time 100–200 μL was plated on LB-agar plates containing 100 μg/mL ampicillin and incubated 12–16 h at 37 °C.
3
Plasmid Construction using Stable E. coli
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We used E. coli strain NEB stable (New England Biolab, MA, USA) that is expected to reduce unexpected recombination in repeat sequences for the plasmid construction. Platinum Gate TALEN kit was obtained from Addgene (#1000000043, Cambridge, MA, USA).
4
Plasmid Construction for Yeast Genome Editing
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ODNs used in this study are listed in Supplementary Table S3 . All ODNs for plasmid construction were purchased from Sigma-Aldrich Japan (Tokyo, Japan) and Eurofins Genomics K. K. (Tokyo, Japan). The four backbone vectors for genome editing were constructed using the seamless cloning with HiFi DNA Assembly (E2621, New England Biolabs, Ipswich, MA, USA). Restriction enzymes used for plasmid construction were purchased from New England Biolabs. PCR fragments used for plasmid construction were amplified by Q5 DNA polymerase (M0491, New England Biolabs) according to manufacturer’s instruction. Escherichia coli competent cells NEB 5-alpha (C2987, New England Biolabs), NEB Stable (C3040, New England Biolabs), or Champion DH5α high (CC5202, SMOBIO Technology, Hsinchu City, Taiwan) were used for transformation to amplify and extract plasmids. Plasmids were extracted by FastGene Plasmid Mini Kit (FG-90502, Nippon Genetics, Tokyo, Japan). Plasmids used in this study are listed in Supplementary Table S2 . The DNA sequence files of the backbone vectors for genome editing are available on our repository at GitHub (https://github.com/poccopen/Genome_editing_plasmid_for_budding_yeast ).
5
Engineered PB-TA-ERP2-mKate2-BRD4S Construct
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We engineered the PB-TA-ERP2-mKate2-BRD4S construct from two addgene clones (65378 [55 (link)] and 80477 [56 (link)]) and pmKate2-H2B (Evrogen, Russia). Overlap extension PCR was performed to amplify the mKate2 insert from the mKate2-H2B plasmid while adding the attB1 adapter and linker sequence. Another round of overlap extension PCR was performed to amplify the BRD4S insert from GFP-BRD4 while adding the attB2 adapter and linker sequence. A final round of fusion PCR was performed to fuse the fragments containing mKate2 and BRD4 to create an insert containing mKate2-linker-BRD4S. The primers used for the overlap extension PCR are listed in S3 Table . A BP reaction using BP Clonase II (Invitrogen, USA) was performed to clone the insert into pDONR221 (Invitrogen), creating the entry clone pENTR221-mKate2-BRD4S. The entry clone was amplified using NEB-Stable (New England Biolabs, USA). An LR reaction using LR Clonase II (Invitrogen) was performed with the destination vector PB-TA-ERP2 [56 (link)] to create the final expression vector.
6
Protein Expression in E. coli
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All purified proteins were produced in BL21 (DE3) pLysS Escherichia coli cells (Novagen) and expression induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 18 hours at 18°C. Amplification of nonviral mammalian expression plasmids was performed in GC10 competent cells (Genesee) and grown at 37°C. Amplification of viral vectors was performed in either Stbl3 (Invitrogen) or NEB Stable (New England Biolabs) competent cells and grown at 30°C.
7
Cloning RAP2.4pro:RAP2.4-venusYFP Construct
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Cloning of RAP2.4pro:RAP2.4-venusYFP was done according to standard In-fusion cloning procedures (Takara In-Fusion Snap Assembly Master Mix #638948), using the linearized pGWB601 vector. Primers used for this cloning can be found in Supplemental Table S1 . The Escherichia coli strains NEB10 (New England Biolabs #C3019H) or NEB stable (New England Biolabs #C3040H; CRISPR constructs) were used for cloning. Electrocompetent Agrobacterium tumefaciens C58C1 RifR (pMP90) or GV3101 RifR bacterial cells (i.e. a cured nopaline strain commonly used for N. benthamiana infiltration) were used for electroporation and N. benthamiana infiltration.
8
Site-directed mutagenesis of ZIKV C protein
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Clustal Omega was used to generate multiple sequence alignments of flavivirus C protein and M protein regions [70 (link)]. Subsequently, Jalview software (University of Dundee, Scotland) was used to analyze and annotate the alignments [71 (link)]. Surface exposed residues of C α3 from the ZIKV C crystal structure (PDB: 5YGH) were selected for substitution to alanine using site-directed mutagenesis using Phusion DNA polymerase (New England Biolabs, #MO5305) and primers shown in S1 Table (IDT DNA Inc.). The reactions were treated with DpnI (New England Biolabs, R0176) followed by transformation of NEB Stable (New England Biolabs) E. coli cells and selection in Lysogeny Broth (LB) agar plates containing 100 ug/ml Ampicillin. Plasmid DNA was purified from isolated colonies using the EconoSpin spin column miniprep kit (Epoch, #2160–250) and sequenced to confirm the mutations at the Huck Genomics Core facility at Pennsylvania State University.
9
Constructing Integrative Plasmids with Yeast Markers
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Plasmids in this work were constructed using pRS40B as a starting point [13 (link)]. The backbone of pRS40B (Figure 1 ) carries sequences relevant for transformation and maintenance in E. coli (bla, ori) and other common features (LacZα, MCS, f1 ori), as well as a TEF promoter and terminator originating from Eremothecium gossypii, known to be able to drive gene expression in various yeast species [14 (link)]. Under the control of the TEF promoter, dominant antibiotic resistance markers needed for selection of yeast transformants and plasmid maintenance in yeast cells were cloned. Markers were codon-optimized to be functional across the Saccharomycotina subphylum (Section 2.3 ) and synthesized de novo. All steps in the construction of integrative plasmids plasmid construction are shown in detail in Figure 1 . DNA manipulations and restriction cloning were performed as in Sambrook (2001). Restriction and modification enzymes were used according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA). Competent cells of E. coli (NEB Stable, New England Biolabs, Ipswich, MA, USA) were prepared and electroporated as in Miller and Nickoloff (1995). Primers were synthesized by Metabion (Planegg/Steinkirchen, Germany). PCR was performed with Q5 polymerase (New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s instructions.
10
Plasmid Construction and Validation
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