Total RNA samples of the following strains and conditions were analyzed: PaSod1-Gfp and PaSod3H26L-Gfp grown in CM- medium without or with 60 µM paraquat, respectively. The cDNA was synthesized from 480 ng total RNA using the iScript kit (Bio-Rad, 170–8891) and was used for qRT-PCR reaction (iQ SybrGreen SuperMix; Bio-Rad, 170–8880). Three biological and 3 technical replicates were analyzed for each strain and each condition. Oligonucleotides (Biomers, Ulm, Germany) used were: Sod1–1 (5′ CAT CAC TGG CCA TGA TGC 3′), Sod1–2 (5′ GCT TGA TGA GGT TGT CGG 3′), Sod3H26L-1 (5′ GAG CCT AAG GGA GAC CTC 3′), Sod3H26L-2 (5′ CTT GCT CAC CAT GGC GTC 3′) Porin-rt-for (5′ TCTCCTCCGGCAGCCTTG 3′) and Porin-rt-rev (5′ GAGGGTGTCGGCAAGTTC 3′). For each gene, the PCR efficiency was determined according to Pfaffl et al.71 (link) The relative expression level (normalized to the level of the PaPorin transcript) was calculated according to Servos et al.72 (link)
Oligonucleotides
Manufactured by Biomers
Sourced in Germany
Oligonucleotides are short, synthetic DNA or RNA molecules that are used in various applications in molecular biology and biotechnology. They are typically composed of 10-100 nucleotides and are designed to serve as probes, primers, or templates for various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Iscript kit, by Bio-Rad (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
11 mercaptoundecyl tetra ethylene glycol, by Merck Group (1 mentions)
Bstui, by New England Biolabs (1 mentions)
Gold enhancement kit, by Nanoprobes (1 mentions)
O 2 mercaptoethyl o methyl hexa ethylene glycol, by Merck Group (1 mentions)
Platinum taq, by Thermo Fisher Scientific (1 mentions)
Dntps, by Roche (1 mentions)
Chromo4 instrument, by Bio-Rad (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Taq 2x master mix, by New England Biolabs (1 mentions)
Hela genomic dna, by New England Biolabs (1 mentions)
Aekta purifier, by GE Healthcare (1 mentions)
S200 10 300 gl column, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by New England Biolabs (1 mentions)
Vent exo dna polymerase, by New England Biolabs (1 mentions)
Human colon total rna, by Thermo Fisher Scientific (1 mentions)
Milli q purification system, by Merck Group (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
13 protocols using oligonucleotides
1
Quantitative Analysis of Sod Genes
2
Nanoparticle-based Molecular Assay
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All oligonucleotides are purchased from Biomers or IDT. Gold nanoparticles are purchased from BB International, silver enhancement kit from Invitrogen and gold enhancement kit from Nanoprobes. All enzymes are purchased from Fermentas except for BstUI which is purchased from New England Biolabs and Tth ligase that is purchased from Genecraft. O-(2-Mercaptoethyl)-O′-methylhexa(ethylene glycol) and (11-mercaptoundecyl)tetra(ethylene glycol) are purchased from Sigma-Aldrich.
3
Real-Time PCR Protocol with KlenTaq Variants
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Oligonucleotides were purchased from Biomers or Metabion, HeLa genomic DNA and Taq 2x master mix was bought from New England Biolabs, dNTPs were either from Roche or Fermentas, Phusion DNA polymerase was purchased from Thermo Scientific, Platinum Taq and AmpliTaq Gold DNA polymerases from Life Technologies, DNase I, SphI, and HindIII from Fermentas, the Gel Extraction and EpiTect MSP kit from Qiagen and used according to their manuals. KlenTaq and its respective mutants were recombinantly expressed in E. coli BL21 (DE3) and purified with Ni-IDA as previously described[63] (link). Enzyme purity and quantity were determined by SDS-PAGE using an albumin standard dilution curve. KlenTaq variants were stored in 50 mM Tris-HCl (pH 9.2), 16 mM (NH4)2SO4, 0.1% Tween20, 2.5 mM MgCl2, 50% glycerol at −20°C. For real-time PCR a Chromo4 instrument from Bio-Rad or a Roche LightCycler 96 or 480 system was used. SYBR green I was purchased from Fluka. PCR was performed in the GeneAmp PCR System 9700 from Applied Biosystems. Genomic DNA samples were bought from NIBSC (Prothrombin Mutation G20210A, Human gDNA, 1st International Genetic Reference Panel 2005 - WHO International Standard or Reference Reagent Product Number 05/130).
4
Synthesis of PMOXA-PDMS-PMOXA Copolymer
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PMOXA15-PDMS68-PMOXA15 (MW/MN = 1.23; MW: mass-average molecular mass; MN: number-average molecular mass) was purchased from Polymer Source Inc. (Dorval, Canada). Enzymes used for DNA work were obtained from New England Biolabs (Frankfurt, Germany). Oligonucleotides were purchased from biomers.net (Ulm, Germany) and Eurofins Genomics (Ebersberg, Germany). All other chemicals were of analytical grade from various suppliers.
5
Designing Stopper DNA Oligomers
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Guidelines for the design of stopper DNA oligomers (named "stoppers" henceforth irrespective of whether they worked efficiently or not) were the same as that for PCR primers. 81 They were designed to avoid secondary structures (with a minimum of 4 base pairs assumed for stable hairpin formation) and self-dimerization (≥5 self-complementary bases). 82 The melting temperatures for RNA/DNA hybrids were estimated using the nearest-neighbor two-state-model and thermodynamic parameters. 83 Salt correction was performed. 84, 85 Overhangs for toehold-release of 8 nts were found to be optimal for rapid strand displacement. 86 Different from those stoppers used with the two TMV-derived RNA scaffolds, wt-RNA and RNA 2253 (see Table 1 and Fig. 1 for details), all stoppers applied with hRNA constructs addressed different non-TMV sequence portions (see Fig. S1 †). Identical toehold overhangs of all DNA oligomers, and their complementary portions in the different release ("fuel") oligomers were used in all the evaluated experiments. Oligonucleotides purified by high performance liquid chromatography were bought from Biomers (Ulm, Germany).
6
Reconstitution of TBP-NC2-Mot1 Complex
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DNA and single protein components were mixed and incubated in a stepwise manner at 4°C. TBP was first added to the TATA box-containing DNA in excess. This was followed by the addition of NC2 and Mot1/Mot1NTD. Finally, the sample was centrifuged and loaded onto a S200 10/300 GL column connected to AEKTA purifier (GE Healthcare). 20 mM MES pH 6.5, 60 mM KCl, 5 mM MgCl2 and 2 mM DTT or 20 mM HEPES pH 8.2, 60 mM KCl, 5 mM MgCl2 and 2 mM DTT were used for the crystallization or EM and CX-MS analyses, respectively. Oligonucleotides were ordered from Biomers, Germany.
7
Fluorescent Oligonucleotide Hybridization
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Oligonucleotides were purchased from Biomers.net GmbH (Ulm, Germany) or Metabion AG (Steinkirchen, Germany). The sequences of the substrates used are (bold letters indicate ribonucleotides):
Each 6-carboxyfluorescein (FAM) 5′-labeled primer was mixed to the complementary template oligonucleotide at 1:1 (M/M) ratio in the presence of 150 mM HEPES–KOH pH 7.4, 500 mM KCl, 10 mM MgCl2 and 250 mM NH4Ac, heated at 95°C for 5 min and then slowly cooled down at room temperature.
Each 6-carboxyfluorescein (FAM) 5′-labeled primer was mixed to the complementary template oligonucleotide at 1:1 (M/M) ratio in the presence of 150 mM HEPES–KOH pH 7.4, 500 mM KCl, 10 mM MgCl2 and 250 mM NH4Ac, heated at 95°C for 5 min and then slowly cooled down at room temperature.
8
Oligonucleotides Synthesis and Purification
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Oligonucleotides (templates and synthetic microRNAs) were purchased from Biomers (Germany). The sequences were purified by high-performance liquid chromatography (HPLC) and checked by matrix-assisted laser desorption/ionization mass spectrometry. Templates were designed according to the rules previously described (23 , 25 (link)). All template sequences (aT, pT, and rT) were protected from the degradation by the exonuclease by 5′ phosphorothioate backbone modifications. A 3′ blocking moiety (phosphate group for aT, pT, and cT and quencher for rT) was used to avoid nonspecific polymerization. Table S1 recapitulates all the sequences used throughout the study. Nb.BsmI and Nt.BstNBI nicking enzymes, Vent(exo-) DNA polymerase, and bovine serum albumin (BSA) were purchased from New England Biolabs (NEB). A 10-fold dilution of Nt.BstNBI was prepared by dissolving the stock enzyme in diluent A (NEB) supplemented with 0.1% Triton X-100. The exonuclease ttRecJ was expressed and purified by chromatography according to a previously published protocol (47 (link)). The enzyme was stored at 1.53 μM in diluent A + 0.1% Triton X-100. All the proteins were stored at −20°C. Human colon total RNA (Thermo Fisher Scientific) was aliquoted at 13 μg/ml and stored at −20°C.
9
Nucleosome DNA Amplification Protocol
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Unless specified otherwise, ultrapure water was used as obtained from a Milli-Q purification system (Merck). All chemicals were purchased from Sigma Aldrich/Merck, VWR, Carl Roth, abcr, Acros organics, TCI or Fluka in analytical or molecular biology grade unless stated otherwise. Chemicals and solvents used for mass spectrometric analyses were purchased in LC-MS grade or UHPLC-MS grade. Oligonucleotides for the PCR of nucleosomal DNA were purchased from biomers.net. The reverse primer was purchased HPLC purified and carrying a 5′-desthiobiotin modification, the forward primer was purchased without modifications and RP-cartridge purified. Oligonucleotides for the double strand DNA ligation assay were purchased from IDT with PAGE purification by the manufacturer. Oligonucleotide sequences were as indicated in Supplementary Table S1 .
10
Magnetic Nanoparticles for Biomedical Applications
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The core–shell MNPs used in
this study were purchased from Micromod Partikeltechnologie, Germany.
The MNPs had a core of 75–80 wt % magnetite, which was encapsulated
in cross-linked hydroxyethyl starch, with surface streptavidin (product
code 10-19-102). These particles had a diameter of around 100 nm and
were suspended in phosphate-buffered saline (PBS) at a concentration
of 10 mg/mL. The theory behind the dynamic magnetic properties of
the NPs is outlined in supporting theory in theSupporting Information .
All oligonucleotides were purchased
from Biomers, Ulm, Germany, and all reagents were purchased from Thermo
Fisher Scientific, Waltham, MA, USA, unless otherwise specified. The
oligonucleotides used in this work are listed inTable 2 .
this study were purchased from Micromod Partikeltechnologie, Germany.
The MNPs had a core of 75–80 wt % magnetite, which was encapsulated
in cross-linked hydroxyethyl starch, with surface streptavidin (product
code 10-19-102). These particles had a diameter of around 100 nm and
were suspended in phosphate-buffered saline (PBS) at a concentration
of 10 mg/mL. The theory behind the dynamic magnetic properties of
the NPs is outlined in supporting theory in the
All oligonucleotides were purchased
from Biomers, Ulm, Germany, and all reagents were purchased from Thermo
Fisher Scientific, Waltham, MA, USA, unless otherwise specified. The
oligonucleotides used in this work are listed in
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