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[求助]
反转录失败，求助已有1人参与
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| 跑胶验证了提取的RNA没有问题，用试剂盒反转录，一批里面总有失败的，就很绝望。也不清楚到底是哪里出了问题 |
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2楼2021-03-17 11:18:27
3楼2021-04-07 09:45:50
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你可以试一下这个方法 First-Strand cDNA Synthesis Using SuperScript™ II RT A 20-μL reaction volume can be used for 1 ng–5 μg of total RNA or 1–500 ng of mRNA. 1. Add the following components to a nuclease-free microcentrifuge tube: Oligo(dT)12-18 (500 μg/mL) or 1 μL 50–250 ng random primers or 2 pmole gene-specific primer (GSP) 1 ng to 5 μg total RNA or x μL 1–500 ng of mRNA 1 μL dNTP Mix (10 mM each) 1 μL Sterile, distilled water to 12 μL 2. Heat mixture to 65°C for 5 min and quick chill on ice. Collect the contents of the tube by brief centrifugation and add: 5X First-Strand Buffer 4 μL 0.1 M DTT 2 μL RNaseOUT™ (40 units/μL) (optional)* 1 μL *RNaseOUT™ (Cat. No. 10777-019) is required if using <50 ng starting RNA. 3. Mix contents of the tube gently. If you are using oligo(dT)12-18 or GSP, incubate at 42°C for 2 min. If you are using random primers, incubate at 25°C for 2 min. 4. Add 1 μL (200 units) of SuperScript™ II RT and mix by pipetting gently up and down. If you are using less than 1 ng of RNA, reduce the amount of SuperScript™ II RT to 0.25 μL (50 units) and add sterile, distilled water to a 20 μL final volume. If you are using random primers, incubate tube at 25°C for 10 min. 5. Incubate at 42°C for 50 min. 6. Inactivate the reaction by heating at 70°C for 15 min. --------来着易基因的回答 |
4楼2021-08-26 17:08:17