pET28b-His10Smt3-csm5Δ46 was constructed via inverse PCR using primers L274/L275 (Supplementary file 2 ) and template pET28b-His10Smt3-csm5 (Walker et al., 2017 (link)). PCR products were digested with DpnI (New England Biolabs) as indicated by the manufacturer and purified using the EZNA Cycle Pure Kit. Purified PCR products were then 5′-phosphorylated with T4 Polynucleotide Kinase for 1 hr at 37°C and circularized with T4 DNA Ligase overnight at room temperature using buffers and instructions provided by the manufacturer. Ligated pET28b-His10Smt3-csm5Δ46 constructs were introduced into E. coli DH5α via chemical transformation. Three transformants were selected, screened, and confirmed using PCR amplification and DNA sequencing with primers T7P/T7T. Confirmed plasmids were purified using the EZNA Plasmid DNA Mini Kit and introduced into E. coli BL21 (DE3) via chemical transformation for protein purification. Three transformants were selected and reconfirmed using PCR amplification and DNA sequencing with primers T7P/T7T (Supplementary file 2 ).
Ezna cycle pure kit
Manufactured by New England Biolabs
Sourced in United States
The EZNA Cycle Pure Kit is a DNA purification product designed to efficiently purify PCR products and other DNA fragments from reaction mixtures. It utilizes a simple spin column-based procedure to remove unwanted primers, nucleotides, and other contaminants, allowing for the recovery of high-quality DNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Dpni, by New England Biolabs (2 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Gateway cloning, by Thermo Fisher Scientific (1 mentions)
Neb monarch pcr dna cleanup kit, by New England Biolabs (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
3 protocols using ezna cycle pure kit
1
Construction of pET28b-His10Smt3-csm5Δ46 Mutant
2
Constructing pET28b-His10Smt3-rnjA/B Plasmids
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pET28b-His10Smt3-rnjA and pET28b-His10Smt3-rnjB were constructed using a two-piece Gibson assembly. Briefly, inserts were obtained by amplifying rnjA and rnjB from the genome of S. epidermidis RP62a and amplifying the backbone from a pET28b-His10Smt3 template using primers L030-L033 (for the rnjB construct) and L042-L045 (for the rnjA construct) (Supplementary file 1 ). PCR products were purified using the EZNA Cycle Pure Kit and Gibson assembled. pET28b-His10Smt3-drnjA, encoding a catalytically-dead variant of RNase J1, was constructed via inverse PCR using primers L055 and L056. PCR products were digested with DpnI (New England Biolabs, MA, USA) and purified using the EZNA Cycle Pure Kit. Purified PCR products were 5’- phosphorylated by incubating with T4 Polynucleotide Kinase at 37°C for 30 min, and then circularized by incubating with T4 DNA Ligase (New England Biolabs, MA, USA) overnight at room temperature. Assembled/ligated constructs were introduced into E. coli DH5α by chemical transformation. Four transformants for each construct were selected and confirmed to have the plasmid using PCR and DNA sequencing with primers T7P and T7T. Confirmed plasmids were purified using the EZNA Plasmid DNA Mini Kit and introduced into E. coli BL21 (DE3) for protein purification.
3
Maize Auxin Signaling Pathway Construction
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Plasmid and primer design was performed in Benchling. Maize Aux/IAA and ARF sequences were obtained from the Grassius Database or synthesized by Integrated DNA Technologies. The maize REL2 fragment was cloned from plasmids generated by Liu et al. (2019) . The maize TIR1/AFB gene sequences were obtained from Paula McSteen. These sequences were ordered from Integrated DNA Technologies with codon optimization for Saccharomyces cerevisiae and then cloned into pCR-BLUNT plasmid using the Zero Blunt TOPO PCR Cloning Kit (James et al., 2000) . The ZmIAA genes were inserted into pGP4GY plasmids (Havens et al., 2012) via Gateway cloning (Life Technologies) or by Gibson cloning (Gibson et al., 2009) . ZmARF27 was cloned into the pGP8G vector by Gibson cloning. Auxin receptors (TIR1 and AFB2s) were cloned into pGP8GF plasmid containing a 3X-FLAG-6X-HIS tandem affinity purification tag. Each PCR amplification was performed using Q5 High-Fidelity DNA Polymerase (New England Biolabs), and the products were purified using the EZNA Cycle Pure Kit or the NEB Monarch PCR & DNA Cleanup Kit and confirmed by sequencing (MCLab or Genewiz). All plasmids used in this research are listed in Supplemental Table S2, and a subset of these have been made available at Addgene.
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