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、dNTPs Mix（以dUTP替代dTTP)、抗体修饰热启动酶（Anstart Taq DNA polymerase）、UDG酶（Uracil-DNA Glycosylase）和MgCl2溶液，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Anstart Taq 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℃、2.5min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
uAnstart Taq DNA polymerase（5U/μl）
uUDG（2U/μl）
udN（U）TP（20/40mM，A:C:G:U=1:1:1:2）
u5×Taq Buffer（Mg2+ Plus）
使用方法
1.PCR 反应体系的设置
a.溶解并混匀 PCR 反应所需的各种溶液，并放置于冰浴上或冰盒内。建议反应 PCR 液体分装使用， 避免反复冻融。
参考下表设置 PCR 反应，建议 PCR 反应体系的配置在冰浴或在冰盒上进行：
|
试剂
|
体积
|
终浓度
|
|
Anstart Taq DNA polymerase（5U/μl）
|
0.5μl
|
—
|
|
UDG（2U/μl）
|
0.25μl
|
—
|
|
dN（U）TP（20/40mM，A:C:G:U=1:1:1:2）
|
0.5μl
|
0.2/0.4mM
|
|
5×Taq Buffer（Mg2+ Plus）
|
10μl
|
1×
|
|
Primer-probe Mix
|
3μl ※
|
—
|
|
模板
|
20μl ※
|
—
|
|
超纯水
|
Up to 50μl
|
—
|
|
总体积
|
50μl
|
—
|
※ 注：引物、探针以及模板的用量可自行调整。
用移液器轻轻吹打混匀或轻微 Vortex 混匀，室温离心数秒，使液体积聚于管底。
各设置好的 PCR 反应管置于 PCR 仪上，开始 PCR 反应。
2. PCR反应参数的设置
|
步骤
|
温度
|
时间
|
循环数
|
|
1
|
50℃
|
2min
|
1
|
|
2
|
95℃
|
2.5min
|
1
|
|
3
|
94℃
|
15s
|
40※
|
|
4
|
55℃
|
40s（收集荧光）
|
※注：Anstart Taq DNA Polymer的热激时间2min30sec到15min都可以，所以第二步的95℃、2.5min（UDG失活，热启动酶热激）的时间可以根据实验要求进行设置，使用时时间从2.5min到15min设置都可以，比较灵活。另收集荧光设置为两步或者三步可以根据实验设置自行设定。
Product manual
This reagent provides a simple, sensitive and anti-pollution PCR detection system. The core reagents include optimized buffer, dNTPs Mix (dUTP instead of dTTP), antibody modification hot-start enzyme (Anstart Taq DNA polymerase), UDG enzyme (Uracil-DNA Glycosylase) and MgCl2 solution. Users only need to add an appropriate amount of Primers and probes or fluorescent dyes can be used for fluorescent PCR detection. Adding UDG before the PCR reaction can degrade PCR products containing uracil without any influence on templates without uracil, and can selectively hydrolyze PCR products containing uracil. The addition of Anstart Taq 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 bases 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. Subsequently, a heat treatment at 95°C for 2.5 min is performed 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
uAnstart Taq DNA polymerase (5U/μl)
uUDG (2U/μl)
udN(U)TP(20/40mM, A:C:G:U=1:1:1:2)
u5×Taq Buffer (Mg2+ Plus)
Instructions
1. PCR reaction system settings
a. Dissolve and mix the various solutions required for the PCR reaction, and place them on an ice bath or in an ice box. It is recommended that the reaction PCR liquid be used in aliquots to avoid repeated freezing and thawing.
Refer to the following table to set up the PCR reaction. It is recommended to configure the PCR reaction system in an ice bath or on an ice box:
|
Reagent
|
Volume
|
Final concentration
|
|
Anstart Taq DNA polymerase（5U/μl）
|
0.5μl
|
—
|
|
UDG（2U/μl）
|
0.25μl
|
—
|
|
dN（U）TP（20/40mM，A:C:G:U=1:1:1:2）
|
0.5μl
|
0.2/0.4mM
|
|
5×Taq Buffer（Mg2+ Plus）
|
10μl
|
1×
|
|
Primer-probe Mix
|
3μl ※
|
—
|
|
Template
|
20μl ※
|
—
|
|
Ultra-pure water
|
Up to 50μl
|
—
|
|
Total capacity
|
50μl
|
—
|
※ Note: The amount of primers, probes and templates can be adjusted by yourself.
Use a pipette to mix gently or gently Vortex to mix, and centrifuge for a few seconds at room temperature to allow the liquid to accumulate at the bottom of the tube.
Place each set of PCR reaction tubes on the PCR machine to start the PCR reaction.
2. Setting of PCR reaction parameters
|
Step
|
Temperature
|
Time
|
Number of cycles
|
|
1
|
50℃
|
2min
|
1
|
|
2
|
95℃
|
2.5min
|
1
|
|
3
|
94℃
|
15s
|
40※
|
|
4
|
55℃
|
40s（Collect fluorescence）
|
※Note: The heat shock time of Anstart Taq DNA Polymer can be 2min30sec to 15min, so the time of 95℃, 2.5min (UDG inactivation, hot start enzyme heat shock) in the second step can be set according to the experimental requirements. It can be set from 2.5min to 15min, which is more flexible. In addition, the two-step or three-step fluorescence collection setting can be set according to the experimental settings.
