Venous blood in serum-tubes and EDTA-tubes were used to determine metabolic parameters and blood cell counts in routine diagnostics. Blood lactate levels were measured with the lactate analyser Biosen 5130 (EKF Diagnostics, Magdeburg, Germany). Respiratory gas exchange analysis (spiroergometry) was carried out using Bluecherry (Geratherm Respiratory GmbH, Bad Kissingen, Germany), and individual anaerobic threshold (IAT) was determined at a net increase of lactate concentration of 1.0 mmol/l above lactate concentration at lactate threshold [21 ]. For cfDNA analysis, capillary blood was centrifuged 2 min at 1,600 x g at 4°C, and nucleic acid concentration was measured in diluted plasma (1:10 in H2O) by direct quantitative real-time PCR of the L1PA2-repeat as previously described [22 ], with slight modifications. The 90 bp L1PA2 fragment was amplified using CFX384 BioRad cycler with 2 min 98°C, followed by 10 s 95°C and 10 s 64°C for 35 cycles. 2 µl of diluted plasma were mixed with 13 µl of Mastermix [final concentrations: Velocity Polymerase 0.6 u, 1.2x Hifi Buffer (Bioline, Luckenwalde, Germany), 0.15x SYBR Green, 0.001x FITC, 0.3 mM dNTPs, 0.15 µM primers] for triplicates in 5 µl final volume.
Velocity polymerase
Manufactured by Meridian Bioscience
Sourced in Germany
Velocity Polymerase is a high-performance DNA polymerase used for PCR amplification. It offers rapid, robust, and accurate DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Hifi buffer, by Meridian Bioscience (2 mentions)
Sybr green, by Merck Group (2 mentions)
Dntps, by Meridian Bioscience (1 mentions)
Ultrapure dnase rnase free h2o, by Thermo Fisher Scientific (1 mentions)
Cfx384, by Bio-Rad (1 mentions)
Genejuice, by Merck Group (1 mentions)
Ampure xp pcr beads, by Beckman Coulter (1 mentions)
Miseq system, by Illumina (1 mentions)
Icycler myiq detection system, by Bio-Rad (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
5 protocols using velocity polymerase
1
Venous Blood Analysis for Metabolic Parameters
2
Quantification of cfDNA in Plasma
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The cfDNA concentrations were determined as described by Neuberger et al. [22 (link)]. Ahead of the measurements, the linearity, limit of quantification, and limit of detection of the assay were determined. The qPCR assay amplifies DNA in unpurified plasma, which is diluted 1:10 in UltraPure DNase/RNase-Free H2O (Invitrogen, Waltham, MA), targeting a 90 bp fragment of human long interspersing nuclear elements (LINEs) of the repetitive L1PA2 family (5′-TGCCGCAATAAACATACGTG-3′ and 5′-GACCCAGCCATCCCATTAC-3′). Briefly, each sample was measured as a technical triplicate with 5 µL final volume containing 0.66 µL of 1:10 diluted plasma, 0.33 µL primer mix (140 nm final concentration of each primer) and 4 µL of qPCR mix with 0.6 U Velocity Polymerase (Bioline, London, UK), 1.2 × Hifi Buffer (Bioline, London, UK), 0.1 × SYBR Green (Sigma, St. Louis, MO, USA), and 0.3 mM dNTPs (Bioline, London, UK). The qPCR reaction was carried out using a CFX384 Bio-Rad (Bio-Rad, Munich, Germany) cycler with a two-step protocol. The cycling conditions were: initial heat activation at 98 °C for 2 min followed by 33 cycles of 95 °C for 10 s and 64 °C for 10 s with a subsequent melting curve from 70 to 95 °C with 0.5 °C increments for 10 s. The operator measuring the samples was blinded and did not know the allocation of the samples into the control or intervention group.
3
Endogenous Tagging of SIK2 with AsCpf1
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Endogenous tagging was performed as described (29 ) and on http://www.pcr-tagging.com . Briefly, the PCR cassette was amplified from pMaCTag-P27 (1 × HA) plasmid in combination with M1_SIK2_fwd and M2_SIK2_AsCpf1_TATV_rev primer using a Velocity polymerase (Bioline) and High-Fidelity (HiFi) buffer (20 mM Tris⋅HCl, pH 8.8, 10 mM [NH4]2SO4, 50 mM KCl, 0.1% [vol/vol] Triton X-100, 0.1 mg/mL BSA, and 2 mM MgCl2) on a gradient PCR cycler. The PCR product was gel purified with GeneJET Gel Extraction Kit. HeLa cells were transiently transfected with 1 µg of the PCR cassette and 1 µg pcDNA3.1-hAsCpf1(TATV) (pY220) using GeneJuice (Merck Millipore) according to the manufacture protocol and selected with 0.5 µg/mL Puromycin 72 h posttransfection.
4
Targeted Amplicon Sequencing Workflow
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Tag- and wild type–specific amplicons from cells used in the experiments shown in Fig. 4 were generated from 200 ng gDNA using junction-specific primers (Table S4 ) by a two-step nested PCR with Velocity polymerase (Bioline). The first PCR reaction was performed for 15 cycles with 60°C annealing and 30 s elongation and then purified with AMPure XP PCR beads (Beckman Coulter). The second PCR was performed for 15 cycles for wild type–specific and for 21 cycles for tag-specific amplicons, respectively, using 60°C annealing and 30 s elongation. PCR products were size selected by gel electrophoresis on 2% agarose/TAE and gel extracted by column purification (Macherey-Nagel). Amplicons were paired-end sequenced with 500 cycles on a MiSeq system (Illumina) using the Amplicon-EZ (150–500 bp) service by Genewiz to acquire at minimum 13,123 reads per sample. Paired reads were merged and aligned to the respective expected amplicon references using CRISPResso (v2.0.29; Kleinstiver et al., 2019 (link)) with parameters “cleavage_offset,” 1; and “window_around_sgrna,” 0. Mutations were subsequently quantified using a custom R script excluding primer binding sites in the analysis.
5
Quantification of Plasma cfDNA by Real-Time PCR
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cfDNA concentrations in plasma were quantified by a direct real-time PCR method (Table 1 ). For the determination of test and software variations all templates were measured in triplicates. 6.4 µl of diluted plasma were added as template to 41.6 µl master mix containing 1 U/µl Tego Buffer (Bioline, Luckenwalde, Germany), 0.05 U/µl Velocity Polymerase (Bioline, Luckenwalde, Germany), 0.17× SYBR Green (Sigma-Aldrich Co., Taufkirchen, Germany), 0.001 µM FITC (Sigma-Aldrich Co., Taufkirchen, Germany), 0.6 mM MgCl2 and 0.34 µM each primer. The total volume of reaction mixture per template was 48 µl adequate to three measurements of 15 µl plus pipetting loss. The final plasma volume per well was 0.05 µl. All reactions comprised a triplicate of non-template controls (NTC).
Reactions were carried out in 96-well PCR plates (0.2 ml tube plate, white, Peqlab, Erlangen, Germany) using the iCycler MyIQ Detection System (Biorad, Munich, Germany) for qPCR measurement. Amplification consisted of an initial denaturation for 2 min at 98°C, followed by 35 cycles of melting at 94°C for 10 s, annealing at 64°C for 40 s and extension at 75°C for 10 s. Subsequent qPCR measurements were calibrated to an interplate-calibration template that had been measured several times on plates containing a standard dilution series.
Reactions were carried out in 96-well PCR plates (0.2 ml tube plate, white, Peqlab, Erlangen, Germany) using the iCycler MyIQ Detection System (Biorad, Munich, Germany) for qPCR measurement. Amplification consisted of an initial denaturation for 2 min at 98°C, followed by 35 cycles of melting at 94°C for 10 s, annealing at 64°C for 40 s and extension at 75°C for 10 s. Subsequent qPCR measurements were calibrated to an interplate-calibration template that had been measured several times on plates containing a standard dilution series.
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