RNA was collected following a protocol similar to Ly and colleagues [49 (link)]. Ten L2 or five L4 animals were picked into 1 μL lysis buffer (5 mM Tris-HCl, pH 8.3, 0.5% Triton X-100, 0.5% Tween 20, 0.25 mM EDTA) with 10% proteinase K (Sigma-Aldrich, Saint Louis, MO). Samples were incubated 10 min at 65°C followed by heat inactivation 1 min at 85°C. Samples were then treated 10 min with 1 U DNase I (New England BioLabs, Ipswich, MA) at 37°C, followed by 20 min heat inactivation at 75°C. cDNA was generated using Invitrogen Superscript III First-Strand Synthesis Supermix for qRT-PCR according to manufacturer’s directions (Invitrogen, Carlsbad, CA). Samples were diluted to 30 μL with diethylpyrocarbonate-treated water before use. qPCR was performed with SYBR GreenER qPCR SuperMix for iCycler Instrument (Invitrogen, Carlsbad, CA) in an iCycler (Bio-Rad, Hercules, CA) using 10 sec 55°C for annealing and 1 min 60°C for elongation. Primer sequences for myo-3 and reference gene ama-1, coding for the largest subunit of RNA polymerase II, are provided in Table 1 . ΔΔCt method was used for quantitation.
Sybr greener qpcr supermix for icycler instrument
Manufactured by Thermo Fisher Scientific
The SYBR GreenER qPCR SuperMix for iCycler Instrument is a ready-to-use real-time PCR reagent designed for quantitative gene expression analysis on the iCycler platform. It contains SYBR Green I dye, required PCR components, and a hot-start Taq DNA polymerase.
Automatically generated - may contain errors
2 protocols using sybr greener qpcr supermix for icycler instrument
1
RNA Extraction and qPCR Analysis
2
RNA Extraction and qPCR Quantification
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RNA was collected following a protocol similar to Ly and colleagues [49] (link). Ten L2 or five L4 animals were picked into 1 µL lysis buffer (5 mM Tris-HCl, pH 8.3, 0.5% Triton X-100, 0.5% Tween 20, 0.25 mM EDTA) with 10% proteinase K (Sigma-Aldrich, Saint Louis, MO).
Samples were incubated 10 min at 65˚C followed by heat inactivation 1 min at 85˚C. Samples were then treated 10 min with 1 U DNase I (New England BioLabs, Ipswich, MA) at 37˚C, followed by 20 min heat inactivation at 75˚C. cDNA was generated using Invitrogen Superscript III First-Strand Synthesis Supermix for qRT-PCR according to manufacturer's directions (Invitrogen, Carlsbad, CA). Samples were diluted to 30 µL with diethylpyrocarbonate-treated water before use. qPCR was performed with SYBR GreenER qPCR SuperMix for iCycler Instrument (Invitrogen, Carlsbad, CA) in an iCycler (Bio-Rad, Hercules, CA) using 10 sec 55˚C for annealing and 1 min 60˚C for elongation. Primer sequences for myo-3 and reference gene (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted September 21, 2020. ; https://doi.org/10.1101/2020.09.21.306985 doi: bioRxiv preprint ama-1, coding for the largest subunit of RNA polymerase II, are provided in Table 1. DDCt method was used for quantitation.
Samples were incubated 10 min at 65˚C followed by heat inactivation 1 min at 85˚C. Samples were then treated 10 min with 1 U DNase I (New England BioLabs, Ipswich, MA) at 37˚C, followed by 20 min heat inactivation at 75˚C. cDNA was generated using Invitrogen Superscript III First-Strand Synthesis Supermix for qRT-PCR according to manufacturer's directions (Invitrogen, Carlsbad, CA). Samples were diluted to 30 µL with diethylpyrocarbonate-treated water before use. qPCR was performed with SYBR GreenER qPCR SuperMix for iCycler Instrument (Invitrogen, Carlsbad, CA) in an iCycler (Bio-Rad, Hercules, CA) using 10 sec 55˚C for annealing and 1 min 60˚C for elongation. Primer sequences for myo-3 and reference gene (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted September 21, 2020. ; https://doi.org/10.1101/2020.09.21.306985 doi: bioRxiv preprint ama-1, coding for the largest subunit of RNA polymerase II, are provided in Table 1. DDCt method was used for quantitation.
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