Genomic DNA was extracted from peripheral blood samples using the G-DEX IIb genomic DNA extraction kit (iNtRon). The relative amount of G-rich single-stranded telomeric DNA was estimated by in-gel hybridization, as previously described (Lamm et al., 2009 (link)). Duplicated samples were electrophoresed in the same gel (0.7% agarose) and, after gel drying, hybridized separately with a C-rich [(TAACCC)3] and G-rich [(AGGGTT)3] probes. The two parts of the gel were washed side-by-side under the same conditions and exposed together to film. Exonuclease I treatment was performed overnight at 37°C with 200 U Exonuclease I (Thermo Fisher Scientific) per 5 µg DNA in 500 µl, followed by ethanol precipitation. The hybridization signal intensity for each lane was determined before and after denaturation using Image J (National Institutes of Health). Native hybridization signal was divided by the denatured signal and normalized to the value calculated for the control samples (set as 1). Mean telomere length was calculated using Matelo (Sagie et al., 2014 (link)).
Exonuclease 1
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Lithuania, Sao Tome and Principe
Exonuclease I is an enzyme that catalyzes the removal of single-stranded DNA from the 3' end. It has a high specificity for single-stranded DNA and is commonly used in molecular biology applications to remove unwanted single-stranded DNA.
Automatically generated - may contain errors
Lab products found in correlation
Fastap thermosensitive alkaline phosphatase, by Thermo Fisher Scientific (26 mentions)
Shrimp alkaline phosphatase, by Thermo Fisher Scientific (20 mentions)
T4 dna ligase, by Thermo Fisher Scientific (10 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (9 mentions)
Fastap alkaline phosphatase, by Thermo Fisher Scientific (9 mentions)
Fastap, by Thermo Fisher Scientific (9 mentions)
Dnase 1, by Thermo Fisher Scientific (7 mentions)
Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (6 mentions)
Alkaline phosphatase, by Thermo Fisher Scientific (6 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (6 mentions)
Abi 3100 dna analyzer, by Thermo Fisher Scientific (4 mentions)
3500 genetic analyzer, by Thermo Fisher Scientific (4 mentions)
Exonuclease 3, by Thermo Fisher Scientific (4 mentions)
3500xl genetic analyzer, by Thermo Fisher Scientific (4 mentions)
Bigdye xterminator purification kit, by Thermo Fisher Scientific (3 mentions)
Hotstartaq dna polymerase, by Qiagen (3 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (3 mentions)
Pcr buffer, by Qiagen (3 mentions)
Bigdye terminator v 3.1 chemistry, by Thermo Fisher Scientific (2 mentions)
Abi prism 3130xl analyzer, by Thermo Fisher Scientific (2 mentions)
111 protocols using exonuclease 1
1
Telomeric DNA Quantification in Peripheral Blood
2
Genomic DNA Hydrolysis Protocol
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Genomic DNA was extracted from the embryonic tissues of the broilers using a classical phenol/chloroform/isoamyl alcohol [25:24:1(v/v/v)] protocol. Residual RNA was treated with RNAses A and T1 (Thermo Fisher Scientific, USA) at final concentrations of 80 μg/mL and 1500 U/mL, respectively, at 37°C for 1 h. The DNA was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and stored at -80°C until analysis.
An improved procedure was established and performed for DNA hydrolysis [17 (link)–19 (link)]. Briefly, a 150-μL enzymolysis system was prepared as follows: approximately 20 μg of tissue DNA, one-tenth volume of 0.1 M ammonium acetate (pH 7.5), 10 μL of DNAse 1 (Thermo Fisher Scientific, USA), 15 μL of 10× DNAse 1 buffer (MgCl2), 10 μL of FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific, USA), 15 μL of 10× Alkaline Phosphatase Buffer, 5 μL of Exonuclease 1 (Thermo Fisher Scientific, USA) and 15 μL of 10× Exonuclease 1 Buffer were mixed, and double-distilled water was added to 150 μL. The mixture was then incubated at 37°C for 4–5 h. The results of agarose gel electrophoresis showed that the genomic DNA was digested to deoxynucleosides (S1 Fig ). The product was filtered through a 0.22-μm nylon membrane filter (Alltech, Deerfield, IL) and stored at -20°C until analysis.
An improved procedure was established and performed for DNA hydrolysis [17 (link)–19 (link)]. Briefly, a 150-μL enzymolysis system was prepared as follows: approximately 20 μg of tissue DNA, one-tenth volume of 0.1 M ammonium acetate (pH 7.5), 10 μL of DNAse 1 (Thermo Fisher Scientific, USA), 15 μL of 10× DNAse 1 buffer (MgCl2), 10 μL of FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific, USA), 15 μL of 10× Alkaline Phosphatase Buffer, 5 μL of Exonuclease 1 (Thermo Fisher Scientific, USA) and 15 μL of 10× Exonuclease 1 Buffer were mixed, and double-distilled water was added to 150 μL. The mixture was then incubated at 37°C for 4–5 h. The results of agarose gel electrophoresis showed that the genomic DNA was digested to deoxynucleosides (
3
Molecular Analysis of Meningococcal and Pneumococcal Infections
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The PCR was performed in 25 µl of a reaction mixture containing 2 µl of isolated
DNA, 10 pmol of each primer, 4 nmol of each deoxynucleotide, 1.5 U of Taq DNA
Polymerase (Sigma-Aldrich, USA), 1 × PCR reaction buffer (containing 15 mM
MgCl2; Sigma-Aldrich, USA) and additionally 25 mmol MgCl2. PCR
products were further purified with thermosensitive Exonuclease I and FastAP
Alkaline Phosphatase (Fermentas, Thermo Fisher Scientific, USA) and sequenced
with BigDye® Terminator v3.1 Cycle Sequencing Kit on an ABI Prism 3130XL
Analyzer (Applied Biosystems, Foster City, CA, USA) according to the
manufacturers’ protocols.
The sequences were compared between cases and healthy family members and the
general population separately for N. meningitidis and
S. pneumoniae cases.
The study was performed with the approval of the Poznan Medical University
Ethical Committee and a written informed consent was obtained from all of the
parents.
DNA, 10 pmol of each primer, 4 nmol of each deoxynucleotide, 1.5 U of Taq DNA
Polymerase (Sigma-Aldrich, USA), 1 × PCR reaction buffer (containing 15 mM
MgCl2; Sigma-Aldrich, USA) and additionally 25 mmol MgCl2. PCR
products were further purified with thermosensitive Exonuclease I and FastAP
Alkaline Phosphatase (Fermentas, Thermo Fisher Scientific, USA) and sequenced
with BigDye® Terminator v3.1 Cycle Sequencing Kit on an ABI Prism 3130XL
Analyzer (Applied Biosystems, Foster City, CA, USA) according to the
manufacturers’ protocols.
The sequences were compared between cases and healthy family members and the
general population separately for N. meningitidis and
S. pneumoniae cases.
The study was performed with the approval of the Poznan Medical University
Ethical Committee and a written informed consent was obtained from all of the
parents.
4
Mutation Analysis via Sequencing
5
Mutation Analysis via Sequencing
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Primers were designed using the Primer3 program and tested for specificity using NIH BLAST software. PCR products were treated with Exonuclease I (Fermentas) and Shrimp Alkaline Phosphatase (USB Corporation) and sequenced using BigDye terminator cycle sequencing Kit v.3.1 on an ABI 3100 DNA analyzer (Applied Biosystems). Sequence data was analyzed by Sequencher 4.9 (Gene Codes) to test segregation of the mutation with the disorder under a recessive mode of inheritance, taking advantage of all informative meioses in each family.
6
Brassica Napus Genotyping Protocol
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Standard molecular biology techniques were performed as described
[46 ]. Oligonucleotides were purchased from Eurofins MWG Operon (Ebersberg, Germany) and sequencing was carried out at the IPK Gatersleben (Germany).
PCR amplifications were carried out in reaction volumes of 30 μl that contained 1 × DreamTaq™ buffer, 250 μM dNTP, 30 pmol of each primer and 1 U of DreamTaq® DNA Polymerase (Fermentas, St. Leon Rot, Germany). Twenty ng of Brassica napus var. Express template DNA were used for PCR amplification. Single BAC clones were assayed for the presence or absence of a particular amplicon by transferring single bacterial colonies to PCR tubes containing all necessary reagents.
PCR samples were preheated for ten min at 95°C and then subjected to 35 PCR cycles. Each cycle consisted of three steps; 30 s at 94°C, 30 s at 60°C, and 60 s at 72°C. After incubation for five min at 72°C the reactions were cooled down to 15°C. Five-μl aliquots were resolved on agarose gels in 1 × TBE.
Prior to sequencing five-μl aliquots of the amplification products were treated with Exonuclease I and FastAP™ Thermosensitive Alkaline Phosphatase as described by the manufacturer (Fermentas, St. Leon Rot, Germany).
[46 ]. Oligonucleotides were purchased from Eurofins MWG Operon (Ebersberg, Germany) and sequencing was carried out at the IPK Gatersleben (Germany).
PCR amplifications were carried out in reaction volumes of 30 μl that contained 1 × DreamTaq™ buffer, 250 μM dNTP, 30 pmol of each primer and 1 U of DreamTaq® DNA Polymerase (Fermentas, St. Leon Rot, Germany). Twenty ng of Brassica napus var. Express template DNA were used for PCR amplification. Single BAC clones were assayed for the presence or absence of a particular amplicon by transferring single bacterial colonies to PCR tubes containing all necessary reagents.
PCR samples were preheated for ten min at 95°C and then subjected to 35 PCR cycles. Each cycle consisted of three steps; 30 s at 94°C, 30 s at 60°C, and 60 s at 72°C. After incubation for five min at 72°C the reactions were cooled down to 15°C. Five-μl aliquots were resolved on agarose gels in 1 × TBE.
Prior to sequencing five-μl aliquots of the amplification products were treated with Exonuclease I and FastAP™ Thermosensitive Alkaline Phosphatase as described by the manufacturer (Fermentas, St. Leon Rot, Germany).
7
PCR Amplification and Sequencing Protocol
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Selected fragments were amplified by PCR using Taq polymerase (Invitrogen) in a Biometra T Professional Gradient 96 cycler. Amplification mixtures contained 10 pmol of each primer, PCR buffer (Invitrogen), 1.5 mM MgCl2, 50 ng of genomic DNA, 200 µM dNTPs, 2.5 U Taq polymerase (Invitrogen), and water to a final volume of 25 µl. After denaturing at 94°C for 2 minutes, thermal cycling was performed for 35 cycles at 94°C for 30 seconds, followed by 30 seconds at a temperature set to 5°C less than the melting temperature of the selected primers, followed by 72°C for 30 seconds. Reactions were finished by a 5 minute incubation at 72°C. Amplification products were checked in 1.2% agarose gels stained with ethidium bromide to verify the presence of a single amplification product. Next, an aliquot (10 µl) of the amplification reaction was treated with 1 U of Exonuclease I (Fermentas) and 10 U of Shrimp Alkaline Phosphatase (Fermentas) for 45 minutes at 37°C and then for 30 minutes at 80°C to inactivate these enzymes. Subsequently two sequencing reactions were prepared, each with one of the primers used for the amplification of the product. Sequencing was carried out in an Applied Biosystems 3130 capillary sequencer using a Big-Dye terminator cycle sequencing kit, according to the instructions of the manufacturer.
8
Strand-Specific qPCR for RNA Detection
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Plates were thawed on ice and refrozen at −70°C 3×. RNA was extracted via the PureLink RNA Micro Kit (Life Technologies) according to the manufacturer's instructions. cDNA was synthesized from total RNA using SuperScript III Reverse Transcriptase (Life Technologies) and 125 nM strand-specific RT primer (+strand_RT: 5′-GGCCGTCATGGTGGCGAATAATGTGATGGATCCGGGGGTAGCG-3′; -strand_RT: 5′-GGCCGTCATGGTGGCGAATAACATGGCAGCCCCGGAACAGG-3′) in a 5-µl reaction. Separate RT reactions for positive and negative-strand RNAs were performed for each sample. RT products were treated with 0.5 units of Exonuclease I (Fermentas) to remove excess RT primer prior to qPCR. Strand-specific qPCR was based on a published protocol (Burrill et al., 2013 (link)). cDNAs were analyzed by qPCR using 2× SYBR FAST Master Mix (Kapa Biosystems), 200 nM strand-specific qPCR primer (+strand_For: 5′-CATGGCAGCCCCGGAACAGG-3′; -strand_Rev: 5′-TGTGATGGATCCGGGGGTAGCG-3′), and 200 nM Tag primer (5′-GGCCGTCATGGTGGCGAATAA-3′) in a 10-µl reaction. A 10× dilution series of in vitro transcribed positive- and negative-strand RNA standards was run alongside experimental samples and used to construct a standard curve.
9
SSCP Analysis and Sequence Validation
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All amplicons were subjected to single-strand conformation polymorphism (SSCP) analysis using protocol B [20 (link)]. Amplicons representing distinct banding profiles were selected and treated with exonuclease I and shrimp alkaline phosphatase (Fermentas), according to the manufacturer’s instructions, and then subjected to direct, automated sequencing (BigDye Terminator v.3.1 chemistry, Applied Biosystems, USA) using the primer CCITS2-R. The quality of each sequence was assessed based on the corresponding electropherogram using the program BioEdit, and the sequences determined were compared with known reference sequences using the Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/BLAST ).
10
Genomic DNA Extraction and Sequencing of Arabis saxatilis
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Extraction of total genomic DNA was performed from silica gel-dried leaves using GenElute Plant Genomic DNA Miniprep Kit (Sigma–Aldrich) and following the manufacturer’s instructions. PCR and sequencing of ndhF were performed as described in Rešetnik et al. (2013) (link) using the primers 5F, 989R, 989F, 1703R, 1410F, and 2100R (Beilstein et al., 2006 (link)). After checking amplicons on 1% TBE-agarose gel, they were purified enzymatically using exonuclease I and shrimp alkaline phosphatase (SAP; Fermentas) following the manufacturer’s instructions. Sequencing was carried out at Macrogen Europe using the same primers as for amplification. Single-digest RADseq libraries were prepared using the restriction enzyme PstI (New England Biolabs) and a protocol adapted from Paun et al. (2016) (link) and Záveská et al. (2021) (link). Up to three individuals of each of 63 populations of A. saxatilis were sequenced on Illumina HiSeq at VBCF NGS Unit1 as 100-bp single-end reads.
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