p685 was constructed by inserting HHO1 (from −111 to +812 with respect to the start codon) as a KpnI-PmeI fragment obtained by PCR into pNEB193 (New England Biolabs) cut with the same enzymes. p687 was constructed by replacing the BamHI-StuI HHO1 fragment in p685 with the BamHI-PmeI URA3 fragment from pNEB-URA3 (18 (link)). pRS-ARG1-B (p688) was obtained by insertion of a PCR fragment containing ARG1 and flanking regions, from −1283 to +2936, relative to the start codon, at the NotI site in pRS414 (Stratagene), a CEN ARS TRP1 plasmid (ARG1 and TRP1 are transcribed in opposite directions). YVN381 (MATa can1–100 his3–11 leu2–3,112 lys2Δ trp1–1 ura3–1 hho1Δ::URA3) was constructed by transforming wild type strain JRY4012 (19 (link)) with a KpnI-PmeI digest of p687.
Pneb193
Manufactured by New England Biolabs
Sourced in Japan
PNEB193 is a lab equipment product offered by New England Biolabs. It is a device used for the purification of nucleic acids. The core function of PNEB193 is to facilitate the extraction and isolation of DNA or RNA samples from various biological sources.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using pneb193
1
Constructing HHO1 and URA3 Plasmids
2
Tomato SlIAA9 Gene Mutation Detection
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Genomic DNA was isolated from tomato leaves using Nucleospin Plant II (TaKaRa, Japan), and used for amplification of target sequences by PCR using PrimeSTAR GXL DNA Polymerase (TaKaRa, Japan). An AccI site is located on the SlIAA9 target sequence, and PCR-RFLP was used for detection of the mutation. In PCR-RFLP, PCR fragments were digested with AccI (NEB, Japan) and analyzed by agarose gel electrophoresis. For Sanger sequencing analysis, PCR fragments purified from agarose gels were cloned by the Seamless ligation cloning extract (SLiCE) method (Motohashi, 2015 (link)) into cloning vector pNEB193 (NEB, Japan). All primers used for PCR are listed in Supplementary Table S1 .
3
Construction of Engineered VAI RNA Scaffold
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Ribozyme cDNA constructs designed against NUH↓ cleavage sites in accessible and inaccessible regions (see Table 1 ) were directionally ligated into the Sal I/Pst I sites in pNEB-VAI-hhRz-1 or pNEB-VAI-hhRz-2 vectors. pNEB-VAI-hhRz-1 and pNEB-VAI-hhRz-2 vectors were generated by cloning the gene for VAI as a BssHII-XbaI fragment from pAdVAntage (E1711; Promega) into pNEB193-T7 (modified by us from pNEB193 from New England Biolabs to have a T7 promoter immediately upstream of the multiple cloning site) and then making extensive further modifications.10 (link),35 (link) Modified stem-loop structures (designed using secondary structure analysis) were added as a series of adapters. Details on the construction of the pNEB-VAI-hhRz-1 plasmid, also known as pUC-VAL, was previously described.17 (link) Details on the construction of the pNEB-VAI-hhRz-2 construct scaffold (pPrislei) are presented (Supplementary Materials ). All constructs were confirmed by DNA sequencing. Because there is a strong intragenic RNA-Pol-III promoter (A, B boxes) in the VAI sequence, both in vitro transcription (with upstream T7 promoter) and in cellula transcription can occur from the same plasmid for either type of construct. Expected RNA structures of native VAI RNA and those of the two engineered VAI hhRz scaffold constructs are shown (Fig. 1 ).
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