Transcription-Coupled DNA Labeling Protocol

DNA was transcribed in 100 μl transcription buffer as aforementioned except that DTT was excluded and 1 mM SBED-GMP^{8 (link)} was added to the transcription buffer. After transcription at 37 °C for 1 hour, the samples were irradiated and subjected to primer extension as the previous studies^{1 (link),4 (link)}. DNA was purified by Wizard SV Gel and PCR Clean-Up System (Promega) followed by primer extension with Deep Vent (exo-) (NEB, M0259) and FAM-5’-TGACAGCGATGCGTAGAATCGCTAG. The G ladder was obtained by extension of non-transcribed DNA in the presence of Acy-CTP (NEB, N0460).

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Zhang J.Y., Xia Y., Hao Y.H, & Tan Z. (2020). DNA:RNA hybrid G-quadruplex formation upstream of transcription start site. Scientific Reports, 10, 7429.

Publication 2020

Wizard SV Gel and PCR Clean-Up System Deep Vent (exo-) Acy-CTP

Buffer Primer Promega Transcription

Corresponding Organization :

Other organizations : Institute of Zoology, Chinese Academy of Sciences

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Variable analysis

independent variables

The presence or absence of DTT (dithiothreitol) in the transcription buffer
The addition of 1 mM SBED-GMP to the transcription buffer

dependent variables

Primer extension after transcription and irradiation

control variables

Transcription buffer composition (except for DTT and SBED-GMP)
Transcription conditions (37 °C, 1 hour)
Primer extension protocol (as in previous studies)

controls

Positive control: Primer extension of non-transcribed DNA in the presence of Acy-CTP to obtain a G ladder

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