Plasmids expressing pegRNAs and epegRNAs were cloned either by Gibson assembly, Golden Gate assembly using either a previously described custom acceptor plasmid14 (link) or newly designed custom acceptor plasmids that contain trimmed evopreQ1 or mpknot (the use of which is described in Supplemental Note 1 ), or synthesized and cloned by Twist Biosciences. Plasmids expressing sgRNAs were cloned via Gibson or USER assembly. DNA amplification was accomplished by PCR with Phusion U or High Fidelity Phusion Green Hot Start II (New England Biolabs). Plasmids expressing pegRNAs were purified using PureYield plasmid miniprep kits (Promega) when transfecting HEK293T cells or Plasmid Plus Midiprep kits (Qiagen) when transfecting other cell types, while plasmids expressing prime editors were purified exclusively using Plasmid Plus Midiprep kits. Plasmids ordered from Twist Biosciences were resuspended in nuclease-free water and used directly. Primers and dsDNA fragments were ordered from Integrated DNA Technologies (IDT). Uncropped agarose and northern blot gels are provided in Supplementary Figs. 16 and 17 .
Plasmid plus midiprep kit
Manufactured by Qiagen
Sourced in United States
The Plasmid Plus Midiprep kits are a set of laboratory equipment used for the purification and isolation of plasmid DNA from bacterial cultures. The kits employ a silica-based membrane technology to efficiently capture and purify plasmid DNA, providing high-quality samples for various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Pureyield plasmid miniprep kit, by Promega (5 mentions)
T7 ligase, by New England Biolabs (2 mentions)
Dh10β cells, by New England Biolabs (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (2 mentions)
Pei max, by Polysciences (2 mentions)
Platinum taq, by Thermo Fisher Scientific (2 mentions)
High fidelity phusion green hot start 2, by New England Biolabs (2 mentions)
Qiaprep spin miniprep kit, by Promega (2 mentions)
Q5 hot start high fidelity 2x master mix, by New England Biolabs (2 mentions)
Phusion u green multiplex pcr master mix, by Thermo Fisher Scientific (2 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Sanger sequencing, by Qiagen (1 mentions)
Hifi assembly cloning kit, by New England Biolabs (1 mentions)
Minelute gel extraction kit, by Qiagen (1 mentions)
14 protocols using plasmid plus midiprep kit
1
Plasmid Cloning for Prime Editing
2
Plasmid Cloning for Prime Editing
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Plasmids expressing pegRNAs and epegRNAs were cloned either by Gibson assembly, Golden Gate assembly using either a previously described custom acceptor plasmid14 (link) or newly designed custom acceptor plasmids that contain trimmed evopreQ1 or mpknot (the use of which is described in Supplemental Note 1 ), or synthesized and cloned by Twist Biosciences. Plasmids expressing sgRNAs were cloned via Gibson or USER assembly. DNA amplification was accomplished by PCR with Phusion U or High Fidelity Phusion Green Hot Start II (New England Biolabs). Plasmids expressing pegRNAs were purified using PureYield plasmid miniprep kits (Promega) when transfecting HEK293T cells or Plasmid Plus Midiprep kits (Qiagen) when transfecting other cell types, while plasmids expressing prime editors were purified exclusively using Plasmid Plus Midiprep kits. Plasmids ordered from Twist Biosciences were resuspended in nuclease-free water and used directly. Primers and dsDNA fragments were ordered from Integrated DNA Technologies (IDT). Uncropped agarose and northern blot gels are provided in Supplementary Figs. 16 and 17 .
3
Subcloning SpCas9 into pTW222 vector
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Post-selection SpCas9 SP pools were subcloned into the pTW222 vector, which encodes nuclease-active SpCas9 variants under control of the arabinose promoter and an sgRNA targeting the HEK3 protospacer sequence on the SacB selection plasmid using an USER enzyme mixture supplemented with T7 ligase (New England Biolabs) as follows: 10 μL 2X T7 ligase buffer, 100,000 U T7 ligase, 1 μL USER enzyme mixture, and 1 μL DpnI was mixed with 0.5 pmol of each DNA fragment, and nuclease-free water was added to a final volume of 20 μL total. The reaction was incubated at 37˚C for 1hr, heated to 80 °C, and slowly (1 °C /sec) cooled to 12 °C, purified using the QIAquick PCR purification kit (Qiagen) according to manufacturer protocol, and transformed into electrocompetent DH10β cells (New England Biolabs). Cells were immediately plated following electroporation on 2xYT media + 1.5% agar supplemented with carbenicillin (50 μg/mL) and incubated at 37 °C for 16–18 h. The resultant colonies were then scraped and plasmids extracted using the Qiagen Plasmid Plus Midiprep Kit (Qiagen) following manufacturer protocol.
4
Subcloning SpCas9 into pTW222 vector
5
Recombinant Protein Expression Vectors
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Parental expression vectors containing
the CDS of an ETE IgG1 mAb, a DTE IgG1 mAb and an ScFv fusion protein
(AstraZeneca, UK) were used for constructing signal peptide varied
plasmids for recombinant protein assays. For each mAb, separate HC
and LC plasmids were provided. Q5 site-directed mutagenesis kits (New
England Biolabs, UK) were used to insert one of 37 signal peptides
directly upstream of each CDS, replacing the control murine Ig HC
signal peptide (MGWSCIILFLVATATGVHS27 (link)).
Transfection-grade plasmid DNA was purified using the QIAGEN plasmid
plus Midiprep kit (QIAGEN, USA).
the CDS of an ETE IgG1 mAb, a DTE IgG1 mAb and an ScFv fusion protein
(AstraZeneca, UK) were used for constructing signal peptide varied
plasmids for recombinant protein assays. For each mAb, separate HC
and LC plasmids were provided. Q5 site-directed mutagenesis kits (New
England Biolabs, UK) were used to insert one of 37 signal peptides
directly upstream of each CDS, replacing the control murine Ig HC
signal peptide (MGWSCIILFLVATATGVHS27 (link)).
Transfection-grade plasmid DNA was purified using the QIAGEN plasmid
plus Midiprep kit (QIAGEN, USA).
6
Plasmid Isolation and Transfection
7
Cloning and Verification of TREM2 Variants
8
Plasmid Isolation and Transfection
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Transformed DH5α cells were grown in 25 to 35 mL Lysogeny Broth (LB) containing ampicillin at 37°C, 225 rpm in a thermal shaker overnight. Cultures were centrifuged (6,000 xg, 15 min at 4°C) and decanted, then subject to DNA plasmid isolation using the Plasmid Plus midiprep kit (QIAGEN), according to the manufacturer’s instructions. For mammalian cell culture transfection, the appropriate amount of DNA plasmid was prepared in a volume of pre-warmed OptiMEM™ reduced-serum medium (ThermoFisher). An equal volume of pre-warmed reduced-serum medium containing PEI MAX (PolySciences) at a 2:1 PEI:DNA ratio was identically prepared. The two solutions were mixed and allowed to incubate at RT for 20 min, after which time the transfection reaction mixture was pipetted drop-wise onto the culture. Culture media was replaced ~4 hr post-transfection with fresh pre-warmed media.
9
Cloning and Vector Preparation Protocol
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We generated all PCR products for cloning with NEB High Fidelity Q5 master mix, and gel extracted and purified PCR products using the Qiagen MinElute gel extraction kit. We created the donor plasmid for homology directed repair using the NEB HiFi assembly cloning kit and the pHD-attP-DsRed vector from flyCRISPR (Gratz et al. 2014 (link)). For plasmid prep, we used Qiagen plasmid plus midi-prep kit and sequenced plasmids with Sanger sequencing (Cornell BRC Genomics Core). We ordered primers for PCR, sequencing, and cloning from IDTDNA (Supplementary Table S2 ).
10
Cloning and Purification of TREM2 Variants
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For immunofluorescence experiments, cDNA corresponding to the full-length TREM2 and D2-TREM2 transcripts was PCR-amplified using the primers corresponding to sequence within exons 1 and 5, described above. For a D2-TREM2 size standard for Western blotting, the D2-TREM2 transcript was cloned with the same exon 1 primer and with the reverse primer corresponding to sequence in the 3'UTR (5' -CCAGCTAAATATGACAGTCTTGGA -3') to preclude an epitope tag. Amplification was performed with Platinum Taq (Invitrogen 10966034) with the following cycling parameters: 2 min at 94°C; 30 s at 94°C, 30 s at 60°C, 2 min at 72°C, 30 cycles; 7 min at 72°C, 25°C hold. All cloning was performed using a pcDNA 3.1-V5/His TOPO-TA cloning kit (Invitrogen K480001) per manufacturer's instructions. Clones were verified by sequencing (ACGT; Wheeling, IL) and grown for midi-scale production and purification using a Qiagen Plasmid Plus Midiprep kit (Qiagen 12943).
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