Hotstart HiTaq Master Mix(含UDG酶)

产品说明

本试剂提供了进行简便、灵敏和防污染的 PCR 检测系统。核心试剂包含了经过优化的缓冲液、化学修饰热启动酶（Hotstart HiTaq DNA polymerase），使用者只需加入适量的引物和探针或荧光染料，即可进行荧光 PCR 检测。在 PCR 反应前加入 UDG 可降解 含有尿嘧啶的 PCR 产物，而对不含尿嘧啶的模板无任何影响，可选择性水解含有尿嘧啶的 PCR 产物。在反应中 Hotstart HiTaq DNA polymerase 的加入使得反应具有极强的特异性和高度灵敏度。

UDG 的作用原理：在 PCR 反应前 50℃、2min 反应中，UDG  酶可水解 PCR 反应液中含有尿嘧啶的 PCR  产物的尿嘧啶碱基和糖磷酸骨架的 N-糖苷键，释放游离尿嘧啶。随后进行 95℃、10min 热处理，在 UDG 酶失活的同时进一步对磷酸骨架水解，从而消除含有尿嘧啶的 PCR 产物的污染。UDG 酶可以作用于单链或双链 DNA，对 RNA 无活性。

产品内容

uHotstart HiTaq Master PCR Mix（25×）

u5×Hotstart HiTaq Buffer( Mg2+  Plus )

使用方法

1. 使用前请注意混匀，然后按下列组份配制 50μl 反应体系的PCR 反应液

※ 注：引物、探针以及模板的用量可自行调整，体系不够请用超纯水补齐。

2. 实验设置

※注：Hotstart  HiTaq  DNA  polymerase 的热激时间 10min，所以第二步的 95℃、10min（UDG 失活，热启动酶热激）的时间需要设置为 10min。另收集荧光设置为两步或者三步可以根据您的实验设置自行设定。

Product manual

This reagent provides a simple, sensitive and anti-contamination PCR detection system. The core reagents include optimized buffers and chemically modified Hotstart HiTaq DNA polymerase. Users only need to add appropriate primers and probes or fluorescent dyes to perform fluorescent PCR detection. Adding UDG before the PCR reaction can degrade PCR products containing uracil without any effect on templates without uracil, and can selectively hydrolyze PCR products containing uracil. The addition of Hotstart HiTaq DNA polymerase in the reaction makes the reaction highly specific and highly sensitive.

The working principle of UDG: In the reaction at 50°C and 2min before the PCR reaction, the UDG enzyme can hydrolyze the uracil base of the PCR product containing uracil in the PCR reaction solution and the N-glycosidic bond of the sugar phosphate backbone to release free uracil. Then heat treatment at 95°C for 10 minutes to further hydrolyze the phosphate backbone while the UDG enzyme is inactivated, thereby eliminating the contamination of PCR products containing uracil. UDG enzymes can act on single-stranded or double-stranded DNA and have no activity on RNA.

Product content

uHotstart HiTaq Master PCR Mix（25×）

u5×Hotstart HiTaq Buffer( Mg2+  Plus )

Instructions

1. Please pay attention to mixing before use, and then prepare the PCR reaction solution of 50μl reaction system according to the following components

※ Note: The amount of primers, probes and templates can be adjusted by yourself. If the system is not enough, please use ultrapure water to make up.

2. Experimental settings

※Note: The heat shock time of Hotstart HiTaq DNA polymerase is 10min, so the time of 95℃, 10min (UDG inactivation, hot start enzyme heat shock) in the second step needs to be set to 10min. In addition, the collection of fluorescence is set to two or three steps, which can be set according to your experimental settings.