Mutational analysis was performed using previously described primers without the MET primer, which was removed from the assay [45 (link)]. The PCR protocol was adapted as below, and performed in a final volume of 10µL, with 50 ng of DNA and 1µM of forward and reverse primers, using 5 µL of the HotStar master mix multiplex (Qiagen, Hilden, Germany) according to the manufacturer´s protocol, with the cycling parameters: 95 °C for 15 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 1 min. 30 s., 72 °C for 1 min and 72 °C for 30 min in a thermal cycler (Veriti, Applied Biosystems, Carlsbad, CA, USA). PCR products were purified with EXO-SAP (Affymetrix, Santa Clara, CA, USA) for 1 h for 37 °C and 15 min for 80 °C. The SNapShot assay was performed as multiplex extension reactions using 5µL of the reaction mix (SNapShot, Applied Biosystems) following the manufacturer´s protocol, with the cycling parameters: 96 °C for 30 s, followed by 35 cycles of 96 °C for 10 s, 50 °C for 5 s, 60 °C for 30 s and 60 °C for 10 min carried out in a thermal cycler (Veriti, Applied Biosystems, Carlsbad, CA, USA). The extension products were separated by electrophoresis in the ABI 3500 XL genetic analyzer (Applied Biosystems/Thermo Fisher Scientific); data were analyzed using the ABI GeneMapper, version 4.0 software (Applied Biosystems/Thermo Fisher Scientific).
Veriti
Manufactured by Thermo Fisher Scientific
Sourced in United States, Australia, Germany, United Kingdom
The Veriti is a thermal cycler designed for PCR amplification. It features a compact and lightweight design, with a capacity of 96 or 384 samples. The Veriti offers precise temperature control and fast ramp rates to ensure efficient and reliable PCR results.
Automatically generated - may contain errors
Lab products found in correlation
Qiaquick pcr purification kit, by Qiagen (6 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (5 mentions)
Dntps, by Thermo Fisher Scientific (4 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (4 mentions)
Isogen, by Nippon Gene (3 mentions)
Hotstar master mix, by Qiagen (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Takara taq dna polymerase, by Takara Bio (3 mentions)
Sybr egel, by Thermo Fisher Scientific (3 mentions)
Mgcl2, by Thermo Fisher Scientific (2 mentions)
Exosap, by Thermo Fisher Scientific (2 mentions)
Abi 3500xl genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Pcr clean up system, by Promega (2 mentions)
Mastermix gotaq, by Promega (2 mentions)
Bigdye terminator cycle sequencing ready reaction kit, by Thermo Fisher Scientific (2 mentions)
Wizard sv gel, by Promega (2 mentions)
Readymix taq pcr reaction mix, by Merck Group (2 mentions)
149 protocols using veriti
1
Multiplex Mutational Analysis Protocol
2
Confirming Viral Sequence Assembly
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In order to confirm the sequences assembled from HTS data, 22 primer pairs were designed on the RNA 1 and RNA 2 HTS-recovered sequences. Total RNA from the IVIA 49.3 grapevine was used as a template. All RT-PCR reactions were performed on the Applied Biosystems Veriti™ thermal cycler (Applied Biosystems, Foster City, CA, USA) using AgPath-ID One-step RT-PCR kit (Ambion Inc., Austin, TX, USA) on a total volume of 25 µL, containing 3 µL of total RNA as template and 0.5 μM of each primer. RT-PCR protocol consisted of one step of 45 °C for 30 min and 95 °C for 10 min, followed by 45 cycles of amplification (95 °C for 30 s, 49–55 °C for 30 s and 60 °C for 1 min). Amplicons were directly sequenced by Sanger, and sequences were aligned using Mega X software [29 (link)].
3
Rapid HLA Typing for DQ2 and DQ8
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HLA typing for DQ2 and HQ8 genotypes was performed using rapid polymerase chain reaction, sequence specific primer (PCR-SSP) method. Primer sequences for HLA DQA1*0501, DQB1*0201, DQA1*0201, DQB1*0202, DQA1*0301 and DQB1*0301 were taken from literature.21 -23 (link, link) Reactions were performed in 25 µl of volume with minor modification in the protocol described in the literature and run over Veriti (Applied Biosystems, CA, USA) thermal cycler. Amplified products were run over 3% agarose gel electrophoresis and visualized on Gel Doc EZ system using Image Lab software, version 5.0 (BioRad, CA, USA).
4
PCR Amplification and Fragment Analysis
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A 35-cycle PCR amplification was carried out in an (Applied Biosystems™ Veriti™, Waltham, MA, USA) 96-Well Thermal Cycler, as follows: 94 °C for 4 min (initial denaturation); 94 °C for 2 min (cyclic denaturation); primer-specific temperature, in °C, for 1 min (annealing); 72 °C for 2 min (cyclic extension); 72 °C for 10 min (final extension); and holding step at 4 °C for unlimited time. The final volume of each sample was 13 μL, containing 2 μL DNA (5 ng/μL), 1.50 μL 10X Buffer (NH4SO4), 1.5 μL MgCl2 (25 mM), 1.5 μL dNTPs (2 mM), 1 μL primer (R + F) (5 μM) and 0.12 μL Taq-DNA polymerase (5 U/μL) (Invitrogen, Carlsbad, CA, USA).
The PCR products were diluted at a ratio of 6 μL sample to 18 μL buffer E of kit DNF 900, and subjected to capillary electrophoresis (Fragment Analyzer, AATI, Santa Clara, CA, USA), where amplified fragments between 35 and 500 bp long were separated at a resolution of approximately 2 bp. Each run lasted 2 h 20 min at 8 kW.
The PCR products were diluted at a ratio of 6 μL sample to 18 μL buffer E of kit DNF 900, and subjected to capillary electrophoresis (Fragment Analyzer, AATI, Santa Clara, CA, USA), where amplified fragments between 35 and 500 bp long were separated at a resolution of approximately 2 bp. Each run lasted 2 h 20 min at 8 kW.
5
Calibrant DNA Sequencing Protocol
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Sequencing reactions for calibrant samples (C1 through C5) were prepared using the following 20 μL reaction mixture: 4–5 ng of template DNA, 1X GeneAmp PCR Gold buffer (Thermo Fisher), 1.5 mM MgCl2 (Thermo Fisher), 1 U AmpliTaq Gold Polymerase (Thermo Fisher), 0.15 μM unlabeled forward and reverse primers (see S6 Table ), 0.16 mg/mL non-acetylated BSA (Invitrogen), 200 μM dNTPs (Invitrogen), and PCR grade water. The SeqAmp program was run on the Veriti (Applied Biosystems) with the following parameters: denaturation for 10 min at 95 °C, amplification for 35 cycles of 1 min at 94 °C, 1 min at 55–60 °C (annealing temperature specific to individual primers), and 1 min at 72 °C, extension for 45 min at 60 °C, followed by a hold at 25 °C.
6
Molecular Detection of Antibiotic Resistance Genes
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Bacterial DNA was extracted by thermal shock (95 °C/20 min) using PBS 1×. Next, the tubes were centrifuged (10,000 rpm/20 min), and the supernatants were separated in new tubes. PCR conditions were the following: 10× PCR Buffer (New England BioLabs, Ipswich, MA, USA) containing 2 mM of MgCl2, 0.2 mM of each of the dNTPs (Invitrogen, Waltham, MA, USA), 10 pmol of each oligonucleotide (Table 5 ), 1.5 U of Taq polymerase (BioLabs, USA), and 3 uL of DNA.
The amplification was carried out using the following program: 1 cycle of 5 min at 95 °C, 35 cycles of 50 s at 95 °C, 60 s at 55 °C, and 50 s at 68 °C, followed by 1 final cycle of 10 min at 68 °C (Veriti, Applied Biosystems, Waltham, MA, USA); Tm for blaIMP was 52 °C, Tm blaoxa-48, blaVIM, blaGES, blaNDM, and blaKPC was 55 °C; Tm for mcr-1 and mcr-2 was 54 °C.
PCR products were separated in a 1% agarose gel at 120 volts for 40 min, and SYBRGreen (Invitrogen, USA) was used such as DNA intercalant. Bands were visualized in Gel Doc XR+ (BioRad, Hercules, CA, USA) with Image Lab Software version 6.1 (BioRad, Hercules, CA, USA).
The amplification was carried out using the following program: 1 cycle of 5 min at 95 °C, 35 cycles of 50 s at 95 °C, 60 s at 55 °C, and 50 s at 68 °C, followed by 1 final cycle of 10 min at 68 °C (Veriti, Applied Biosystems, Waltham, MA, USA); Tm for blaIMP was 52 °C, Tm blaoxa-48, blaVIM, blaGES, blaNDM, and blaKPC was 55 °C; Tm for mcr-1 and mcr-2 was 54 °C.
PCR products were separated in a 1% agarose gel at 120 volts for 40 min, and SYBRGreen (Invitrogen, USA) was used such as DNA intercalant. Bands were visualized in Gel Doc XR+ (BioRad, Hercules, CA, USA) with Image Lab Software version 6.1 (BioRad, Hercules, CA, USA).
7
RNA Extraction and Reverse Transcription
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Total cellular RNA was extracted from the left hind limb bone of the mice using Isogen (Nippon Gene, Toyama, Japan), according to the manufacturer’s instructions. Total RNA was reverse-transcribed using the Transcriptor First Strand cDNA Synthesis kit (Roche Applied Science, Penzberg, Germany) with a DNA thermal cycler (Veriti; Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s protocol.
8
PCR Detection of SARS-CoV-2 Genome Deletion
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To detect the 382-nucleotide deletion in the SARS-CoV-2 genome, we used two specific PCR primers (forward 5ʹ-TGTTAGAGGTACAACAGTACTTT-3ʹ; reverse 5ʹ-GGTAGTAGAAATACCATCTTGGA-3ʹ) flanking the deleted region.5 (link) For samples with high cycle threshold (Ct) values, hemi-nested PCR was done with a second forward primer (5ʹ-TGTTTATAACACTTTGCTTCACA-3ʹ) and the same reverse primer as before. The PCR mixture contained the cDNA primers (10 μM each), 10 × Pfu reaction buffer (Promega, Madison, WI, USA), Pfu DNA polymerase (Promega), and 10 mM dNTP mix (Thermo Scientific, Waltham, MA, USA). PCR was done in a thermal cycler (Applied Biosystems Veriti, Foster City, CA, USA) with the following conditions: 95°C for 2 min, followed by 35 cycles at 95°C for 1 min, 52°C for 30 sec, and 72°C for 1 min; and a final extension at 72°C for 10 min. Deletions in the PCR products were visualised with use of a QIAxcel DNA screening cartridge on QIAxcel Advanced capillary electrophoresis system (Qiagen, Hilden, Germany).
9
PCR Analysis of p16 Microsatellite Marker
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For PCR analysis, p16 microsatellite marker DS91747 with the sequence of forward 5’-ATTCAACG AGTGG GATGAAG-3’ and reverse 5’-TCCAGGTTGCTGCAAATGCC-3’ was selected. The PCR product size expected was 130–150 bp.
Ampli Taq red master mix containing 2 mM MgCl2, 0.2 mM of dNTPs and 1 U/μl of Taq DNA polymerase was prepared as reaction mixture. Primer concentration of 0.4 μM and 3 μl of DNA template was added to the mixture. To carry out amplification, a Veriti thermal cycler (Applied Biosystems, CA, USA) was used. After this, thermal cycling conditions were used for running the PCR which was 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 54°C for 1 min and 72°C for 1 min. Finally, 2% agarose gel was prepared and the 15 μl samples were loaded on to the gel which was further subjected to electrophoresis at 80 V for 1 h. The gel was subsequently stained with 0.5 μg/ml ethidium bromide and observed under ultraviolet gel documentation system (Major Science, Saratoga, CA, USA).[14 (link)]
The molecular weight of the band was confirmed by comparing the location with standard molecular weight marker ladder (100–1500 bp).[12 ]
Ampli Taq red master mix containing 2 mM MgCl2, 0.2 mM of dNTPs and 1 U/μl of Taq DNA polymerase was prepared as reaction mixture. Primer concentration of 0.4 μM and 3 μl of DNA template was added to the mixture. To carry out amplification, a Veriti thermal cycler (Applied Biosystems, CA, USA) was used. After this, thermal cycling conditions were used for running the PCR which was 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 54°C for 1 min and 72°C for 1 min. Finally, 2% agarose gel was prepared and the 15 μl samples were loaded on to the gel which was further subjected to electrophoresis at 80 V for 1 h. The gel was subsequently stained with 0.5 μg/ml ethidium bromide and observed under ultraviolet gel documentation system (Major Science, Saratoga, CA, USA).[14 (link)]
The molecular weight of the band was confirmed by comparing the location with standard molecular weight marker ladder (100–1500 bp).[12 ]
10
Reverse Transcription of Total RNAs
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Total RNAs (including miRNAs) were converted into cDNAs using by TaqMan microRNA reverse transcription kit. This kit converts total RNAs into cDNA using specific miRNA primers which are provided with respective Taqman MicroRNA Assays.For each sample, a 15μl reaction was setup on ice having 7 μl of RT master mix (0.25 mM dNTPs, 3.33 U/μL multiscribe RT enzyme, 1X RT buffer, 0.25 U/μL RNAse inhibitor), 3 μl of specific reverse primer and 5 μl of RNA sample. Reaction tubes were incubated at temperature of 16°C for 30 mins, 42°C for 30mins and last at 85°C for 5mins in a Veriti thermal cycler (Applied Biosystems).
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