Oct 02, 2020

Public workspace反转录cDNA

This protocol is a draft, published without a DOI.
  • 1西南大学
  • Zhang Xue
张 雪
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Protocol Citation: 张 雪 2020. 反转录cDNA. protocols.io https://protocols.io/view/cdna-bmyqk7vw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 02, 2020
Last Modified: October 02, 2020
Protocol Integer ID: 42736
Before start
全程RNase-free 枪头！！！
方法A.Ominiscript kit
方法A.Ominiscript kit

Note
RNase-free枪头

10×buffer RT2µl
dNTP mix(5mM) 2µl
oligo-dT primer(10µM)2µl
RNase inhibitor1µl
Ominiscript reverse transcriptase1µl
Template RNA2µg
RNase-free H2Oadd to 20µl

混匀，37°C，1hTemperature37 °C Duration01:00:00

测浓度
方法B. Thermo kit合成cDNA
方法B. Thermo kit合成cDNA
使用8联管便于后续用PCR仪控温
Note
RNase-free枪头

total RNA0.1ng～5µg
Oligo(dT)18 Primer1µl
RNase-free H2Oadd to 12µl

混匀，65°C，5min，此后在冰上操作Temperature65 °C Duration00:05:00


向上述体系继续加入以下组分TemperatureOn ice

5×reaction buffer4µl
RNase inhibitor1µl
10mM dNTP mix2µl
reverse transcriptase1µl

混匀，45°C，1hTemperature45 °C Duration01:00:00

72°C，5minTemperature72 °C Duration00:05:00

测浓度