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PCR amplification of transcription products

PW Phillip Wibisono
YL Yiyong Liu
JS Jingru Sun
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Reverse transcription reactions containing 2 μl of purified RNA and 2 μl of 10 mM HNqPCRrev1 primer in a final volume of 20 μl were performed using the Qiagen Sensiscript RT kit (Qiagen, catalog # 205211) per the manufacturer’s instructions. After the reaction was completed, 1 μl of the reverse transcription mix was transferred to a PCR tube and used for PCR. The standard PCR reaction was performed with the HNqPCRrev1 and HNqPCRfrw1 primers using the Failsafe PCR system with the Premix A (Lucigen, catalog # F599100). Positive and negative PCR control reactions with or without 50 ng of HNDNA or PESDNA, respectively, were also performed. After amplification, the PCR products were analyzed by gel electrophoresis on a 2.0% agarose TAE gel, followed by staining with SYBR safe DNA gel stain (Invitrogen, catalog # S33102), imaging with iBright 1500, and quantification with software ImageJ.

Copyright and License information: The Author(s) ©2020
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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