产品说明
SYBR Green Anstart Taq PCR Mix(2×)是采用SYBR Green I 嵌合荧光法进行Real Time PCR的专用试剂。该试剂中含有Real Time PCR反应的最适浓度SYBR Green I,是一种2×浓度的SYBR Anstart 试剂,进行实验时,PCR反应液的配制十分方便简单。本试剂中使用了抗体修饰的热启动酶(Anstart Taq DNA Polymerase)与Real Time PCR用最适Buffer组合,可以有效抑制非特异性的PCR扩增,大大提高PCR 的扩增效率,可以进行高灵敏度的Real Time PCR扩增反应。
产品内容
SYBR Green Anstart Taq PCR Mix(2×)。
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。
b.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
|
SYBR Green Anstart Taq PCR
|
25μl
|
1×
|
Primer I(10μM)
|
1μl※
|
0.2μM
|
Primer II(10μM)
|
1μl※
|
0.2μM
|
Template DNA
|
10μl※
|
—
|
超纯水
|
Up to 50μl
|
|
总体积
|
50μl
|
|
※ 注:引物以及模板的用量可自行调整,体系不够请用超纯水补齐。
c.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。
d.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)
|
5
|
Dissociation
|
—
|
1
|
注:由于 mix 中使用的 DNA 聚合酶热激时间从 1min 到 15min 都可以,所以第二步的 95 ℃、2.5min 热启动酶热激的时间可以根据实验要求进行设置,使用时间从 2.5min 到 15min 设置都可以,比较灵活。退火温度可根据您的引物 Tm 值适当调整,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
SYBR Green Anstart Taq PCR Mix (2×) is a special reagent for Real Time PCR using SYBR Green I chimeric fluorescence method. This reagent contains the optimum concentration of SYBR Green I for Real Time PCR reaction. It is a 2× concentration of SYBR Anstart reagent. When conducting experiments, the preparation of PCR reaction solution is very convenient and simple. This reagent uses an antibody-modified hot-start enzyme (Anstart Taq DNA Polymerase) combined with the most suitable buffer for Real Time PCR, which can effectively inhibit non-specific PCR amplification, greatly improve PCR amplification efficiency, and perform high-sensitivity Real Time PCR amplification reaction.
Product content
SYBR Green Anstart Taq PCR Mix (2×).
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 to use aliquots of the reaction PCR liquid to avoid repeated freezing and thawing.
b. 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
|
SYBR Green Anstart Taq PCR
|
25μl
|
1×
|
Primer I(10μM)
|
1μl※
|
0.2μM
|
Primer II(10μM)
|
1μl※
|
0.2μM
|
Template DNA
|
10μl※
|
—
|
Ultra-pure water
|
Up to 50μl
|
|
Total capacity
|
50μl
|
|
※ Note: The amount of primer and template can be adjusted by yourself. If the system is insufficient, please use ultrapure water to make up.
c. 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.
d. Place each set of PCR reaction tubes on the PCR machine to start the PCR reaction.
2. PCR reaction parameter setting
Step
|
Temperature
|
Time
|
Number of cycles
|
1
|
50℃
|
2min
|
1
|
2
|
95℃
|
2.5min
|
1
|
3
|
94℃
|
15s
|
40
|
4
|
55℃
|
40s(Collect fluorescence)
|
5
|
Dissociation
|
—
|
1
|
Note: Since the DNA polymerase heat shock time used in the mix can be from 1 min to 15 min, the second step 95 ℃, 2.5 min hot start enzyme heat shock time can be set according to the experimental requirements, the use time is from 2.5 min to It can be set for 15 minutes, which is more flexible. The annealing temperature can be adjusted according to the Tm value of your primers, and the fluorescence collection can be set to two or three steps, which can be set according to your experimental settings.