20 minute PCR Enzymatic Cleanup
Julian Liber
Abstract
A rapid microplate-compatible protocol for cleanup of PCR products prior to Sanger sequencing. Verify that the PCR produces a single band, and retain some PCR product for cleanup and sequencing. Set up at room temperature, and react on the thermocycler.
Mechanism: PCR products contain 1) primers and 2) dNTPs which will interfere with downstream Sanger sequencing reactions. Exonuclease I degrades ssDNA (primers) and calf intestinal phosphatase degrades dNTPs. Functions like NEB #E1050.
Steps
Making the enzyme master mix
Add to a 1.5 mL sterile conical tube
- 760 uL water
- 94 uL 10x rCutSmart buffer
- 96 uL 10x Exonuclease I buffer (NEBuffer r3.1)
- 4 uL Quick CIP (5U/uL) NEB #M0525
- 10 uL Thermolabile Exonuclease I (20U/uL) NEB #M0568
Mix, then aliquot 200 uL into separate tubes. Store at -20C. Good for at least 2 years.
PCR Cleanup
Mix 1.2 uL of master mix with 1.5 uL PCR product in PCR strip tubes or plates.
#尊敬的用户，由于网络监管政策的限制，部分内容暂时无法在本网站直接浏览。我们已经为您准备了相关原始数据和链接，感谢您的理解与支持。
<iframe width="560" height="315" src="https://www.youtube.com/embed/n1-Nze7vYJI" title="YouTube video player" frameborder="0" allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture; web-share" allowfullscreen=""></iframe>
#尊敬的用户，由于网络监管政策的限制，部分内容暂时无法在本网站直接浏览。我们已经为您准备了相关原始数据和链接，感谢您的理解与支持。
<iframe width="560" height="315" src="https://www.youtube.com/embed/n1-Nze7vYJI" title="YouTube video player" frameborder="0" allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture; web-share" allowfullscreen></iframe>
Seal tubes or plates and place on a thermocycler. React at 37ºC for 15 minutes, then 80ºC for 1 minute.
Add the product to Sanger sequencing reactions. 1 uL of product, 1 uL of 10 uM primer, and 10 uL of water per tube. If the band is faint, you can use 2 uL or more of product.
