Primer extension was performed on total RNA (10 μg) using the SuperScript III Reverse Transcriptase (Invitrogen), according to the manufacturer's instructions with the following modifications. RNA samples were annealed with 3 μl of radiolabeled primers at 65°C during 30 min and kept on ice for 1 min. The reverse transcription was done at 55°C during 1 h (50°C for OLEC3926). The cDNA samples were then precipitated with ethanol, resuspended in 5 μl of 1X loading dye and resolved on 10% polyacrylamide/8 M urea/TBE gels. The primers were end-radiolabeled as previously described (28 ) and the AFLP 30–300 bp ladder (Invitrogen) was labeled according the manufacturer's instructions using the T4 Polynucleotide kinase (PNK) (Thermo Fischer Scientific).
T4 polynucleotide kinase pnk
Manufactured by Thermo Fisher Scientific
Sourced in United States
T4 polynucleotide kinase (PNK) is an enzyme that catalyzes the transfer of the gamma-phosphate from ATP to the 5' hydroxyl terminus of DNA, RNA, or oligonucleotides. This enzyme is commonly used in molecular biology applications to label the 5' end of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (8 mentions)
γ 32p atp, by PerkinElmer (3 mentions)
Factor x activated, by Merck Group (2 mentions)
γ 32p atp, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Promega (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
G50 column, by GE Healthcare (1 mentions)
T4 dna ligase buffer, by Thermo Fisher Scientific (1 mentions)
Tetracycline, by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Thermo Fisher Scientific (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Trimethoprim, by Merck Group (1 mentions)
Genesys 10 uv, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Polymyxin b, by Merck Group (1 mentions)
Restriction endonuclease, by Takara Bio (1 mentions)
Taq dna polymerase, by Transgene (1 mentions)
M mlv reverse transcriptase, by Transgene (1 mentions)
Cyclone plus phosphor imager, by PerkinElmer (1 mentions)
17 protocols using t4 polynucleotide kinase pnk
1
Primer Extension RNA Analysis Protocol
2
G-Quadruplex Oligonucleotide Labeling and Folding
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T4 polynucleotide kinase (PNK) (Thermo Fisher Scientific) and γ-32P-ATP were used to 5′ end label 0.5 μM 10A-G4 oligonucleotides or their mutated variants at 37°C for 75 min. Subsequently, 1 μl 0.5 M ethylenediaminetetraacetic acid (EDTA) was added and the reaction was incubated at 78°C for 1 min to inactivate the T4 PNK. Labeled DNA was purified on a G50 column (GE Healthcare).
To fold the G4 oligonucleotides, 20 μl of labeled 0.2 μM 10A-G4 oligonucleotides, or their mutated variants, was mixed with an equal volume 2 × folding buffer (20 mM Tris (pH 7.5) and 200 mM KCl). The reaction was incubated at 95°C for 5 min and allowed to cool down to room temperature for 3 h. The oligonucleotides were loaded on a 10% native polyacrylamide gel containing 50 mM KCl and separated in a cold ice box run at 100 V for 80 min.
To fold the G4 oligonucleotides, 20 μl of labeled 0.2 μM 10A-G4 oligonucleotides, or their mutated variants, was mixed with an equal volume 2 × folding buffer (20 mM Tris (pH 7.5) and 200 mM KCl). The reaction was incubated at 95°C for 5 min and allowed to cool down to room temperature for 3 h. The oligonucleotides were loaded on a 10% native polyacrylamide gel containing 50 mM KCl and separated in a cold ice box run at 100 V for 80 min.
3
Phosphorylation and Proximity Ligation
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The air-dried bead-nuclei mixture was resuspended in 100 μL 1× T4 DNA ligase Buffer with ATP containing 1 U/μL T4 Polynucleotide Kinase (PNK) (Thermo), and incubated at 37 °C for 1 h to phosphorylate ligated bridge adaptors. Following incubation, 90 μL 10× T4 DNA ligase Buffer with ATP, 6 μL T4 DNA ligase (5 U/μL; Thermo), and 804 μL of H2O were added to the reaction mix. In situ proximity ligation was then carried out at room temperature for 4 h.
4
Oligonucleotide-Based Fluorogenic Assay
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The sequences of all synthetic DNA oligonucleotides are provided in S2 Fig. . The fluorogenic substrate FS1, which contains an adenosine ribonucleotide as the cleavage site flanked by a fluorescein-dT and a dabcyl-dT, was purchased from Yale University Keck Facilities. The other DNA oligonucleotides were purchased from Integrated DNA Technologies (IDT). All oligonucleotides were purified by 10% denaturing (8 M urea) polyacrylamide gel electrophoresis (dPAGE) and their concentrations were determined based on the UV absorbance at 260 nm (Genesys UV 10, Thermo Scientific). T4 DNA ligase, T4 polynucleotide kinase (PNK) and ATP were obtained from Thermo Scientific. All the antibiotics (kanamycin, ampicillin, trimethoprim, chloraphenicol, tetracycline and polymyxin B) and other chemicals were purchased from Sigma-Aldrich. The bacteria E. coli K12 MG1655 and Bacillus subtilis 168 are routinely maintained in our laboratory. Listeria monocytogenesis and Staphylococcus aureus were obtained from the Shenzhen Centre for Disease Control and Prevention (Shenzhen, Guangdong province, China). Water used here was double-deionized (ddH2O) and autoclaved.
5
Purification and Activation of MazF Toxin
6
Enzymatic Ligation and Synthesis Protocol
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T4 polynucleotide kinase (PNK), T4 DNA ligase, T7 RNA Polymerase and Bsm DNA Polymerase were purchased from Thermo Scientific. Taq DNA Polymerase and M-MLV reverse transcriptase were purchased from Transgen (Beijing, China). ATP, NTP, dNTP and all of the DNA sequences used in our research were purchased from Sangon Biotech (Shanghai, China) Co., Ltd. [γ-32P]ATP were purchased from Perkin Elmer. Restriction endonuclease was purchased from TaKaRa Biotechnology (Dalian, China) Co., Ltd. Rabbit Reticulocyte Lysate a translation system was purchased from Promega. The molecular weight of the ligation products was measured by the high resolution mass spectrometry (HRMS). The melting temperatures were detected by HITACHI U-1900 spectrophotometer. N-Cyanoimidazole was synthesized by the reaction between cyanogen bromide and imidazole and dissolved in chloroform after purification by filtration and rotary evaporation. Radioactive isotope γ-32P was detected by Cyclone Plus Phosphor Imager (Perkin Elmer).
7
RNA Quantification in H. volcanii
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Total RNA was isolated from H. volcanii cells in the early exponential growth phase (OD650 nm: ∼0,3) with TRIzolTM Reagent (InvitrogenTM, Thermo Fisher Scientific). DNase treatment to remove residual DNA was performed with RQ1 RNase-Free DNase (Promega). Ten micrograms of total RNA was separated by 8% PAGE and transferred to a nylon membrane (Hybond-N+, GE Healthcare). The membrane was hybridized with radioactively labeled oligonucleotide probes. Probes were labeled at the 5′ end by T4-polynucleotide kinase (PNK; Thermo Fisher Scientific) and [γ-32P]-ATP. To quantify the RNA, imaging plates (BAS-MS, Fujifilm) were exposed to radioactivity on the hybridized membranes and analyzed with the FLA-3000 scanner (GE Healthcare; software BASreader 3.14). Signal intensity was measured with ImageJ and set in relation to the signal of 5S rRNA that was detected and used as a loading control. The proportion of RNA is given as a percentage from cells expressing a targeting spacer and those carrying a control plasmid. The amount of RNA in the control strains was set to 100%, and the amounts in strains expressing a targeting spacer were calculated in relation to this value. Northern blot analyses were performed with biological triplicates of the control and CRISPRi strains.
8
Oligonucleotide Synthesis and Purification Protocol
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All oligonucleotides
(Table S1 ) in this study were HPLC-purified
and synthesized by Shanghai Sangon Biological Engineering Technology
and Services Co., Ltd. (Shanghai, China). The secondary structures
of designed sequences were predicted using online bioinformatic software
(NUPACK: Nucleic Acid Package). All DNAs were dissolved in 1×
TE buffer (10 mM Tris, 1 mM EDTA, PH 8.0), and the concentration was
determined by a NanoDrop 2000 (Thermo Fisher Scientific, USA). Adenosine
triphosphate (ATP), T4 DNA ligase, and 10× T4 DNA ligase buffer
were provided by Takara Biotechnology Co., Ltd. (Dalian, China). T4
polynucleotide kinase (PNK) and 10× PNK buffer were obtained
from Thermo Scientific (MA, USA), and phi29 MAX DNA polymerase and
the corresponding reaction buffer (10×) were provided by Vazyme
Biotech Co.,Ltd. (Nanjing, China). A low-molecular-weight DNA ladder
and deoxyribonucleoside 5′-triphosphate (dNTP) mixture were
all supplied by Shanghai Sangon Biological Engineering Technology
and Services Co. Ltd. (Shanghai, China). SYBR Green I was purchased
from Solarbio Biotechnology Co., Ltd. (Beijing, China). All chemicals
were of analytical grade without further purification and dissolved
in ultrapure water produced by the MiNiQ-Direct 8 Laboratory Small
Pure Water System (electrical resistance of 18.25 MΩ cm at 25
°C) that was purchased from Merck (Merck, GER).
(
and synthesized by Shanghai Sangon Biological Engineering Technology
and Services Co., Ltd. (Shanghai, China). The secondary structures
of designed sequences were predicted using online bioinformatic software
(NUPACK: Nucleic Acid Package). All DNAs were dissolved in 1×
TE buffer (10 mM Tris, 1 mM EDTA, PH 8.0), and the concentration was
determined by a NanoDrop 2000 (Thermo Fisher Scientific, USA). Adenosine
triphosphate (ATP), T4 DNA ligase, and 10× T4 DNA ligase buffer
were provided by Takara Biotechnology Co., Ltd. (Dalian, China). T4
polynucleotide kinase (PNK) and 10× PNK buffer were obtained
from Thermo Scientific (MA, USA), and phi29 MAX DNA polymerase and
the corresponding reaction buffer (10×) were provided by Vazyme
Biotech Co.,Ltd. (Nanjing, China). A low-molecular-weight DNA ladder
and deoxyribonucleoside 5′-triphosphate (dNTP) mixture were
all supplied by Shanghai Sangon Biological Engineering Technology
and Services Co. Ltd. (Shanghai, China). SYBR Green I was purchased
from Solarbio Biotechnology Co., Ltd. (Beijing, China). All chemicals
were of analytical grade without further purification and dissolved
in ultrapure water produced by the MiNiQ-Direct 8 Laboratory Small
Pure Water System (electrical resistance of 18.25 MΩ cm at 25
°C) that was purchased from Merck (Merck, GER).
9
Radiolabeling and Nuclease Assays
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One μL of the nucleic acids was treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific, Waltham, MA, USA) in a 10-μL mixture containing 1 μL of 10X Buffer (Thermo Fisher Scientific) at 37 °C for 30 min. Mock treatment using water instead of FastAP was performed as controls (- FastAP). Then, the samples were heated at 90 °C for 10 min. One μL of the heated samples was labeled with γ32P-ATP (PerkinElmer, Waltham, MA, United States) by T4 polynucleotide kinase (PNK, Thermo Scientific) using the forward reaction buffer at 37 °C for 20 min. Then, the samples were analyzed by denaturing polyacrylamide gel electrophoresis. The gel was exposed to a phosphor screen and imaged by a Typhoon 5 laser-scanner (Cytiva, Marlborough, MA, USA).
The labeled nucleic acids were further treated with RNase A (DNase and protease-free, Thermo Scientific, EN0531) or DNase I (RNase-free, Thermo Scientific, EN0521) for 1 h at 37 °C, and analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography as described above.
The labeled nucleic acids were further treated with RNase A (DNase and protease-free, Thermo Scientific, EN0531) or DNase I (RNase-free, Thermo Scientific, EN0521) for 1 h at 37 °C, and analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography as described above.
10
Bacterial Culture Reagents Preparation
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Agar, yeast extract and tryptone broth were from Carl Roth KG (Karlsruhe, Germany). T4 polynucleotide kinase (PNK) was obtained from Thermo Scientific (Darmstadt, Germany). All other chemicals were from Sigma-Aldrich (Steinheim, Germany). Oligonucleotides in triple HPLC-purified quality were purchased from Purimex (Grebenstein, Germany). The corresponding sequences are listed in Supplementary Table S1 .
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