Full-length mtDNA amplification was carried out for short-read and long-read sequencing, and as a part of quality control using Platinum SuperFi II DNA Polymerase (Invitrogen). A forward primer Mt2120F (5′- GGA CAC TAG GAA AAA ACC TTG TAG AGA GAG −3′) and a reverse primer Mt2119R (5′- AAA GAG CTG TTC CTC TTT GGA CTA ACA −3′) were used. All lrPCRs (50 μl) were prepared as follows: 1× SuperFi II buffer, 0.4 mM each of dNTPs, 0.2 μM of each forward and reverse primers, 1× of SuperFi II DNA Polymerase, 0.5 mM MgCl2, and 10–100 ng of individual DNA sample. Cycling conditions were: 94 °C for 1 min; 30 cycles of 98 °C for 10 s, 68 °C for 16 min; 72 °C for 10 min; hold at 4 °C. To visualise amplicons, Ultra Pure Agarose 1% (Invitrogen) gel was prepared in 1× TAE buffer (BIO-RAD). DNA samples were loaded together with Quick-Load 1 kb Extend DNA Ladder (NEB) to aid size determination. Fragments were separated running a gel at 60 V for 2.5–3 h. Amplicons were purified with AMPure XP beads (Agencourt, Beckman Coulter) and eluted in 30 μl nuclease-free water. Concentration was measured using a Qubit fluorometer with the dsDNA Broad Range Assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Quick load 1 kb extend dna ladder
Manufactured by New England Biolabs
Sourced in Germany
The Quick-Load 1 kb Extend DNA Ladder is a pre-stained, ready-to-load DNA molecular weight marker designed for quick and easy visualization of DNA fragment sizes ranging from 100 base pairs to 20,000 base pairs. It is suitable for use in agarose gel electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Dsdna broad range assay kit, by Thermo Fisher Scientific (2 mentions)
Platinum superfi 2 dna polymerase, by Thermo Fisher Scientific (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Ultra pure agarose 1, by Thermo Fisher Scientific (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Purple gel loading dye, by New England Biolabs (1 mentions)
Electrophoresis equipment, by Bio-Rad (1 mentions)
Quick load purple 1 kb dna ladder, by New England Biolabs (1 mentions)
La taq hot start polymerase, by Takara Bio (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
Q5 high fidelity 2x master mix, by New England Biolabs (1 mentions)
3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Takara la taq hot start polymerase, by Takara Bio (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Qubit 4, by Thermo Fisher Scientific (1 mentions)
Genomic tip 500 g, by Qiagen (1 mentions)
8 protocols using quick load 1 kb extend dna ladder
1
Comprehensive mtDNA Amplification for Sequencing
2
Plasmid Size Determination by Gel Electrophoresis
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To determine the size of plasmids, a 0.8% agarose (MIDSCI) gel was run at 120 V for ~40 min on an electrophoresis equipment (Bio‐Rad). Before electrophoresis, extracted plasmids were mixed with the Purple Gel Loading Dye (6×, no SDS; New England Biolabs) and loaded into wells in the agarose gel. Quick‐Load® Purple 1 kb DNA Ladder (New England Biolabs) or Quick‐Load® 1 kb Extend DNA Ladder (New England Biolabs) was used for the reference of DNA length. Gel images were inverted by ImageJ for visualization purpose.
3
Mitochondrial DNA Deletion Detection
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Total DNA was isolated from the apexes of dissected hearts using the QIAamp DNA Mini Kit (QIAGEN, Venlo, The Netherlands) column purification. The DNA isolation protocol provided by the manufacturer was followed and the samples were eluted in 200 µL elution buffer provided in the kit. All samples were stored at 4 °C. Deletions of mitochondrial DNA were detected by a long-range PCR protocol using the TaKaRa LA Taq Hot Start polymerase (Takara, Saint-Germain-en-Laye, France) specifically suitable for the production of longer PCR amplicons with greater accuracy. In order to detect deletions, almost the entire mtDNA was amplified between primers musMT2482F24 (5′-GTTCAACGATTAAAGTCCTACGTG-3′) and musMT1005R24 (5′-CCAGTATGCTTACCTTGTTACGAC-3′), under the following conditions: 95 °C for 2.5 min, 30 cycles of 92 °C for 20 s and 66.8 °C for 5.5 min, and final extension at 72 °C for 10 min. The PCR products were visualized on a 1% agarose gel with Quick-Load 1 kb Extend DNA Ladder (New England Biolabs, Frankfurt/Main, Germany).
4
Evaluating gDNA Quantity, Quality, and Integrity
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gDNA extractions were evaluated for quantity and quality using the Qubit dsDNA BR Assay Kit (Invitrogen, Waltham, MA) and NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, Pittsburgh, PA) following manufacturer’s instructions. gDNA integrity was assessed following 0.6% agarose gel electrophoresis of 100 ng of each sample to visualize the presence of intact, high molecular weight (HMW) DNA. The Quick-Load 1 kb Extend DNA Ladder (New England BioLabs, Ipswich, MA) was used as a molecular marker.
5
Sequencing of O-Antigen Deletion Region
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Primers were design to target the gap of the O-antigen deletion region in EC18 strains (gap-F 5′-TCA AGC ACC GAA TAA CCT -3′ ) and (gap-R 5′-TAC CTG AAG TAC GTA GCC-3′ ). The primers were designed based on the sequence of the two contigs adjacent to the O-antigen deletion region, for which no direct linkage information was available from the genome sequence. As the size of the expected product was not immediately clear from the genome assembly alone, long-range PCR was performed using Q5 High-Fidelity 2X Master Mix (New England Biolabs). PCR was performed as follows; 98°C for 30 sec, 30 cycles of 98°C for 10 sec, 52°C for 30 sec and 72°C for 30 sec, and a final extension at 72°C for 2 min. Electrophoresis in a 1% agarose gel was used to determine the size of the PCR product by running with Quick-Load 1 kb Extend DNA Ladder (New England Biolabs). The DNA sequence of the amplified product was determined by Sanger sequencing at the Plymouth University Systems Biology Centre using an Applied Biosystems 3130 Genetic Analyzer. Sequence data were assembled using CLC Main Workbench v.6.9.1.
6
Mitochondrial DNA Deletion Detection
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Total DNA from left ventricular muscle was isolated through column purification with QIAamp DNA Mini Kit (QIAGEN N.V., Venlo, Netherlands) as described by the manufacturer. Each sample was eluted in 200 μl elution buffer provided with the kit and stored without freezing at 4°C. LR-PCR was used in order to detect mtDNA deletions. For that purpose, almost the entire mtDNA was amplified between primers musMT2482F (5’-GTTCAACGATTAAAGTCCTACGTG-3’ ) and musMT1005R (5’-CCAGTATGCTTACCTTGTTACGAC-3’ ) (first number, 5’-end of the primer; F-forward or R-reverse) by the use of TaKaRa LA Taq Hot Start polymerase (Clontech). The LR-PCR was performed under the following conditions: 95°C for 2.5 min, 30 cycles of 92°C for 20 s and 66.8°C for 5:30 min, and 72°C for 10 min. The PCR products were loaded on a 1% agarose gel with Quick-Load 1 kb Extend DNA Ladder (New England Biolabs, NEB).
7
Genomic DNA Extraction from Dalbergia Species
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Dried seeds of D. cochinchinensis and D. oliveri were collected from the Bolikhamxay, Khamkend, Laos, and Phnom Penh, Cambodia in 2018 by their forestry authorities, respectively. We germinated the seeds in a greenhouse at 30 °C with 16L/8D photoperiod. Leaf tissues were harvested from a selected 1-y-old individual for each species and ground in liquid nitrogen with a mortar and pestle.
High-molecular-weight genomic DNA was extracted from the reference individual with Carlson lysis buffer (100 mM Tris-HCl, pH 9.5, 2% CTAB, 1.4 M NaCl, 1% PEG 8000, 20 mM EDTA) followed by purification using the QIAGEN Genomic-tip 500/G. The quantity and quality of genomic DNA were determined with NanoDrop 2000 (Thermo, Wilmington, United States) and Qubit 4 (Thermo Fisher Scientific, United Kingdom). DNA integrity was preliminary assessed with a 0.4% agarose gel against a NEB Quick-Load® 1 kb Extend DNA Ladder. A DNA sample passed the quality check only when a single band could be mapped near a lambda DNA band (~ 48.5 kb).
High-molecular-weight genomic DNA was extracted from the reference individual with Carlson lysis buffer (100 mM Tris-HCl, pH 9.5, 2% CTAB, 1.4 M NaCl, 1% PEG 8000, 20 mM EDTA) followed by purification using the QIAGEN Genomic-tip 500/G. The quantity and quality of genomic DNA were determined with NanoDrop 2000 (Thermo, Wilmington, United States) and Qubit 4 (Thermo Fisher Scientific, United Kingdom). DNA integrity was preliminary assessed with a 0.4% agarose gel against a NEB Quick-Load® 1 kb Extend DNA Ladder. A DNA sample passed the quality check only when a single band could be mapped near a lambda DNA band (~ 48.5 kb).
8
Amplifying Full-Length mtDNA for Sequencing
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Full-length mtDNA amplification was carried out for short-read and long-read sequencing, and as a part of quality control using Platinum SuperFi II DNA Polymerase (Invitrogen). A forward primer Mt2120F (5′-GGA CAC TAG GAA AAA ACC TTG TAG AGA GAG -3′) and a reverse primer Mt2119R (5′-AAA GAG CTG TTC CTC TTT GGA CTA ACA -3′) were used. All lrPCRs (50 μl) were prepared as follows: 1× SuperFi II buffer, 0.4 mM each of dNTPs, 0.2 μM of each forward and reverse primers, 1× of SuperFi II DNA Polymerase, 0.5 mM MgCl2, and 10-100 ng of individual DNA sample. Cycling conditions were: 94°C for 1 min; 30 cycles of 98°C for 10 s, 68°C for 16 min; 72°C for 10 min; hold at 4°C. To visualize amplicons, Ultra Pure Agarose 1% (Invitrogen) gel was prepared in 1× TAE buffer (BIO-RAD). DNA samples were loaded together with Quick-Load 1 kb Extend DNA Ladder (NEB) to aid size determination.
Fragments were separated running a gel at 60 V for 2.5-3 h. Amplicons were purified with AMPure XP beads (Agencourt, Beckman Coulter) and eluted in 30 μl nuclease-free water. Concentration was measured using a Qubit fluorometer with the dsDNA Broad Range Assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions. incubating at 37ºC for 20 min followed by Quick CIP inactivation at 80ºC for 3 min. For the following Cas9-mtDNA-enrichment procedure each DNA sample was split into aliquots.
Fragments were separated running a gel at 60 V for 2.5-3 h. Amplicons were purified with AMPure XP beads (Agencourt, Beckman Coulter) and eluted in 30 μl nuclease-free water. Concentration was measured using a Qubit fluorometer with the dsDNA Broad Range Assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions. incubating at 37ºC for 20 min followed by Quick CIP inactivation at 80ºC for 3 min. For the following Cas9-mtDNA-enrichment procedure each DNA sample was split into aliquots.
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