PCR reaction volumes were 20µl, comprising 1ng extracted human DNA, 1x Phire buffer, 1mM dNTPs (Fisher Scientific, Loughborough, UK), 150ng/µl BSA (Roche, Burgess Hill, UK), 222nM VKOF primer, 1µM VKOR primer, 500nM P2-JP probe, 500nM VK-UC1, 500nM VK-UC2 and 0.8µl Phire polymerase (Fisher Scientific, Loughborough, UK). The sequences of primers, probes and universal complement oligonucleotides are detailed in Table 3. Following an initial denaturation step (98°C for 1 minute), targets were amplified using 50 cycles comprising denaturation (98°C for 5 seconds) and annealing/extension (65°C for 10 seconds). Melting curve analysis was performed immediately after amplification by briefly denaturing samples (98°C for 1 minute) and cooling (20°C for 1 minute) prior to increasing temperature from 20°C to 60°C in 0.5°C steps.
Phire buffer
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Phire buffer is a high-performance PCR buffer designed for use with the Phire Hot Start II DNA Polymerase. It is formulated to provide efficient and reliable DNA amplification across a wide range of templates and target sizes.
Automatically generated - may contain errors
Lab products found in correlation
Dntps, by Thermo Fisher Scientific (4 mentions)
Phire polymerase, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Roche (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Chelex solution, by Bio-Rad (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Phire hot start 2 dna polymerase, by Thermo Fisher Scientific (1 mentions)
2720 thermal cycler, by Thermo Fisher Scientific (1 mentions)
4 protocols using phire buffer
1
Detecting VKOR Genetic Variants
2
Real-Time PCR Amplification Protocol
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PCR reaction volumes were 20µl, comprising 1ng extracted human DNA, 1x Phire buffer, 1mM dNTPs (Fisher Scientific, Loughborough, UK), 2mM MgCl 2 , 150ng/µl BSA (Roche, Burgess Hill, UK), 5% DMSO (Fisher Scientific, Loughborough, UK), 222nM 1661-F primer, 1µM 1661-R primer, 150nM P2-JP probe, 500nM 1661-UC1, 500nM 1661-UC2 and 0.8µl Phire polymerase (Fisher Scientific, Loughborough, UK). The sequences of primers, probes and universal complement oligonucleotides are detailed in Table 3. Following an initial denaturation step (98°C for 1 minute), targets were amplified using 50 cycles comprising denaturation (98°C for 5 seconds) and annealing/extension (65°C for 10 seconds). Melting curve analysis was performed immediately after amplification by briefly denaturing samples (98°C for 30 seconds) and cooling (35°C for 30 seconds) prior to increasing temperature from 35°C to 60°C in 0.5°C steps.
3
DNA Extraction from Mites and Eggs
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After sampling, we stored adult males individually in 96% ethanol for preservation. Prior to DNA extraction, we evaporated the ethanol, and then transferred the mites into 1.5 ml tubes containing 50 μl of 5% Chelex solution (Bio-Rad Laboratories, Hercules, CA, USA) and 3–4 zirconium beads. We homogenized samples for 20 s using a Precellys24 tissue homogenizer (Bertin Corp, Rockville, MD, USA), and added 2.5 μl of proteinase K (20 mg ml−1) to each sample, followed by incubation at 56 °C for 60 min, then denaturation of the proteinase at 95 °C for 8 min. We centrifuged samples at 14 000 rpm for 2 min, and stored them at -20 °C until amplification. We collected individual inviable eggs using an ethanol- and flame-sterilized pin, and crushed them in 10 μl of 5 × Phire buffer (Thermo Fisher Scientific, Waltham, MA, USA) in a PCR tube. Samples were centrifuged at 4000 rpm for 2 min and then stored at −20 °C. Prior to amplification, we pipetted the buffer-egg mix up and down to facilitate mixing of buffer and sample. To generate control samples for the PCR process, we followed the above processes without transferring a sample into the reaction medium.
4
Total RNA Extraction and RT-PCR Analysis
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Total RNA from EiPSCs and ViPSCs was collected after ten passages (P10) and isolated by standard TRIzol®-chloroform isolation and iso-propanol precipitation. cDNA was derived from the collected mRNA by using SuperScript IV Reverse Transcriptase (SSIV) (ThermoFisher). For RT-PCR 1 μg of RNA and Random Hexamer Primers were used and incubated with SSIV, 0,1 M Dithiothreitol (DTT) and 40 U/μl RNaseOUT (ThermoFisher) for 10 min at 23 °C, 10 min at 50 °C and 10 min at 80 °C. Afterwards samples were stored at −20 °C until further processed. PCR analysis was performed with different primer sets (Tab. S1 ) with Phire Hot Start II DNA Polymerase (ThermoFisher). To perform PCR, a mastermix containing Phire Buffer (ThermoFisher), dNTPs (10 mM) (ThermoFisher) and Phire Hot Start II DNA Polymerase was mixed and divided over different PCR tubes. 1 μl of each primer (10 μM) and 1 μl cDNA was added to the tubes and carefully mixed. Samples were placed in thermocycler (Applied Biosystems™ 2720 Thermal Cycler) and PCR program was initiated (Tab. S2 ).
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