Mouse cDNAs of β2a, β2c, and β2c-short were cloned by V. Flockerzi (Saarland University, Homburg, Germany). For the C-terminal fusion of green fluorescent protein (GFP) to each β2 subunit, the cDNAs encoding β2a, β2c, and β2c-short were amplified by PCR using nTaq DNA polymerase (Enzynomics), TA cloned into T-Easy Vector (Promega), and cloned in pEGFP-N1 vector (Takara Bio Inc.). The primers used for β2-GFP are listed in Table S1. For point and deletion mutants, β2-GFP was amplified by inverse PCR using Pfu Turbo DNA polymerase (Agilent Technologies), plasmid DNA was digested by Dpn I (Agilent Technologies), the PCR product was 5′-phosphorylated by T4 polynucleotide kinase (Enzynomics), and then PCR product was ligated by T4 DNA ligase (New England Biolabs, Inc.). The primers used for mutagenesis are listed in Table S2. The mutants were verified by DNA sequencing.
T4 polynucleotide kinase
Manufactured by Enzynomics
Sourced in Cameroon
T4 polynucleotide kinase is an enzyme that catalyzes the transfer of a phosphate group from ATP to the 5' hydroxyl terminus of DNA or RNA molecules. It is commonly used in molecular biology techniques to label nucleic acids with radioactive or non-radioactive phosphate groups.
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Lab products found in correlation
γ 32p atp, by PerkinElmer (3 mentions)
T4 dna ligase, by Enzynomics (2 mentions)
Labopass gel extraction kit, by Cosmogenetech (2 mentions)
Kod plus neo dna polymerase, by Toyobo (2 mentions)
Sanger sequencing, by Macrogen (2 mentions)
T easy vector, by Promega (1 mentions)
Ntaq dna polymerase, by Enzynomics (1 mentions)
Pegfp n1 vector, by Takara Bio (1 mentions)
Dpni, by Agilent Technologies (1 mentions)
Pfu turbo dna polymerase, by Agilent Technologies (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Bas 1500 imaging system, by Fujifilm (1 mentions)
Ribomax large scale rna production system, by Promega (1 mentions)
Antarctic phosphatase, by New England Biolabs (1 mentions)
Subtilisin, by Merck Group (1 mentions)
Poly di dc, by Merck Group (1 mentions)
Pcr primers, by Cosmogenetech (1 mentions)
Restriction enzymes, by Enzynomics (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
α factor, by GenScript (1 mentions)
8 protocols using t4 polynucleotide kinase
1
Cloning and Tagging of β2 Calcium Channel Subunits
2
Characterization of ToxR Binding to leuO Promoter
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To assess the binding of the ToxR to a cis-acting element of leuO, a 412-bp DNA fragment containing the upstream region of the leuO promoter (nucleotides +60 to −352 with respect to the translation start site of leuO) was PCR-amplified using leuO-F and leuO-R primers. To assess the binding of the ToxR-N to DNA fragment containing the region upstream of leuO including each of a 312-bp wild type fragment, the same fragments with a 5-bp or a 10-bp deletion between the promoter and the fragment with a deletion in the ToxR-binding site, and a 20-bp deletion in the ToxR binding site were amplified using primers lacZ-sleuO-F and leuO_R2. The PCR products were subsequently labeled with [γ-32P]ATP using T4 polynucleotide kinase (Enzynomics, Daejeon, Korea). For gel-mobility shift assays, 10 ng of the labeled DNA fragment was incubated with increasing amounts of purified ToxR (0 to 800 nM) or ToxR-N (0 and 100 nM) in a 20 μl reaction in binding buffer [10 mM Tris-HCl (pH 7.4), 10 mM KCl, 1 mM EDTA, 0.1 mM DTT, 50 μg/ml bovine serum albumin, and 5% glycerol] for 30 min at 30 °C. The reaction was terminated by the addition of 4 μl loading buffer, and samples were resolved on a 6% neutral polyacrylamide gel. The DNA was visualized using the BAS 1500 imaging system (Fujifilm, Tokyo, Japan).
3
Production and Purification of Labeled BC200 RNA
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Template DNAs for in vitro transcription were obtained by PCR-amplification of BC200 RNA or its derivative sequences from plasmids pSUPER-BC200_A6, pSUPER-BC200_A11, pSUEPR-BC200_A14, and pSUPER-BC200_A24 using the primer pair, BC200_T7_F (5′-GAA TTC TAA TAC GAC TCA CTA TAG GCC GGG CGC GGT G-3′) and BC200_R (5′-AAA GGG GGG GGG GGG TTG TTG CTT TG-3′). In vitro transcription was carried out using a RiboMAX Large Scale RNA Production System (Promega, USA). In vitro transcripts were gel-purified. When required, transcripts were 5′-labeled with [γ-32P]ATP (PerkinElmer, USA) using T4 polynucleotide kinase (Enzynomics, Korea) after treatment with Antarctic phosphatase (New England Biolabs, USA).
4
Preparation of Radiolabeled Substrates and ssDNA
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Oligonucleotides used for the preparation of radiolabeled substrates and ssDNA competitor were synthesized and gel‐purified by Bioneer (Daejeon, Korea). [γ‐32P]ATP (3000 Ci·mmol−1) was purchased from Perkin Elmer (Waltham, MA, USA). PCR primers were synthesized by Cosmo Genetech (Seoul, Korea). Subtilisin, MMS, CPT, propidium iodide, and poly(dI‐dC) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). M13mp18 sscDNA was acquired from New England Biolabs (Ipswich, MA, USA). α‐factor was purchased from GenScript (Piscataway, NJ, USA). Restriction enzymes, T4 polynucleotide kinase, and protein size markers were obtained from Enzynomics (Daejeon, Korea).
5
Detailed PCR Experimental Protocol
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All PCR experiments were conducted with KOD Plus neo DNA polymerase (Toyobo, Japan). T4 polynucleotide kinase and T4 DNA ligases were purchased from Enzynomics (South Korea). Plasmids and DNA fragments were purified with LaboPassTM plasmid DNA purification kit mini and LaboPass™ Gel extraction kit (Cosmogenetech, South Korea). Sequences of all DNA constructs in this study were confirmed by Sanger sequencing (Macrogen, South Korea and Bionics, South Korea). Antibiotics (carbenicillin, chloramphenicol, kanamycin), arabinose, and Isopropyl β-d -1-thiogalactopyranoside (IPTG) were purchased from LPS solution (South Korea). Streptomycin was purchased from Sigma Aldrich. Tetracycline was purchased from Bio Basic. Cefotaxime and ceftazidime were purchased from Tokyo chemical industry (Japan). H-p-Chloro-dl -Phe-OH (p-Cl-Phe) was purchased from Bachem (Switzerland).
6
Northern Blot Analysis of RNA
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Northern blot analysis was performed as described previously (21 (link)). Total RNA was fractionated on a 6% polyacrylamide gel containing 7 M urea, and transferred to a Hybond-XL membrane (GE Healthcare). Antisense oligonucleotides were 5′ end labeled with [γ-32P] ATP (PerkinElmer Life Sciences) by T4 polynucleotide kinase (Enzynomics) and used as probes for hybridization. Hybridization was carried out in Rapid-Hyb buffer (GE Healthcare), and samples were incubated at 40°C overnight (for BC200 RNA) or at 42°C for 1 h (for the 5S RNA). The membrane was washed twice (20 min each time) at 25°C in 2× SSC buffer (20 mM sodium phosphate, pH 7.4, 0.3 M NaCl, 2 mM EDTA) containing 0.1% SDS, and twice (20 min each time) in 0.2× SSC buffer containing 0.1% SDS. The membrane was exposed to an imaging plate (Fuji BAS-IP), and the results were analyzed on a phospho-image analyzer (Fuji FLA-7000).
7
Detailed Biomolecular Protocols for Research
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All PCR experiments were conducted with KOD Plus neo DNA polymerase (Toyobo, Japan). T4 polynucleotide kinase and T4 DNA ligases were purchased from Enzynomics (South Korea). Plasmids and DNA fragments were purified with LaboPass™ plasmid DNA purification kit mini, LaboPass™ PCR purification kit, and LaboPass™ Gel extraction kit (Cosmogenetech, South Korea). Sequences of the cloned genes were confirmed by Sanger sequencing (Macrogen, South Korea). Antibiotics (ampicillin, chloramphenicol, kanamycin, and tetracycline), arabinose, and IPTG were purchased from LPS solution (South Korea). Carbon sources were obtained from Sigma-Aldrich (d -tartrate, #147-71-7; erythritol, #149-32-6; sucrose, #57-50-1; ethylene glycol, #29810; l -lyxose, #1949-78-6; 2-deoxy-d -glucose, #205-823-0; d -(+)-cellobiose, #528-50-7) and Acros (monomethyl succinate, #3878-55-5; 2-deoxy-d -ribose, #533-67-5; l -sorbose, #87-79-6).
8
Site-Saturation Mutagenesis of Sniper1 Codon
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For site saturation mutagenesis of the 1,007th codon in the Sniper1 sequence, the pBLC–Sniper1 plasmid was amplified using primers containing NNK (K = G or T) at the appropriate position (forward primer: agtaccccaagctggagagcnnkttcgtgtacggcgactacaagg; reverse primer: tcttgatcagggcggtgcc). PCR products were digested with DpnI (Enzynomics), treated with T4 polynucleotide kinase (Enzynomics) and ligated with T4 ligase (Enzynomics). The resulting product was transformed in DH5alpha cells. After Sanger sequencing of plasmids from 100 randomly selected colonies, variants containing 20 different amino acids at the 1,007th position were identified.
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