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Hifair^? Ⅲ One Step RT-qPCR SYBR Green Kit

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Hifair® Ⅲ One Step RT-qPCR SYBR Green Kit是以RNA為模板進(jìn)行定量PCR反應(yīng)的試劑盒。在實驗的過程中，逆轉(zhuǎn)錄和定量PCR在同一反應(yīng)管中進(jìn)行，簡化了實驗操作，降低了污染的風(fēng)險。本試劑盒以RNA為模板，使用基因特異性引物，利用耐熱Hifair® Ⅲ Reverse Transcriptase高效合成第一鏈cDNA，并以此為模板在UNICON® HotStart Taq DNA Polymerase作用下，配合優(yōu)化的緩沖體系能夠特異靈敏的對RNA模板進(jìn)行精確定量。同時Buffer中額外添加了有效抑制非特異性PCR擴(kuò)增的組分和提高擴(kuò)增能力的促進(jìn)因子，使定量PCR結(jié)果可以在寬廣范圍內(nèi)更加真實有效。

本試劑盒以Kit形式提供，2×Hifair® Ⅲ SG buffer包含優(yōu)化的緩沖體系和dNTPs等，優(yōu)化了有效抑制非特異性PCR擴(kuò)增的因子和提升PCR反應(yīng)擴(kuò)增效率的因子，適用于SYBR Green標(biāo)記的高特異性檢測系統(tǒng)。此外，Hifair® UH Ⅲ Enzymes包含比例優(yōu)化的Hifair® Ⅲ Reverse Transcriptase、RNase inhibitor及UNICON® HotStart Taq DNA Polymerase等。

產(chǎn)品信息

貨號	11143ES50 / 11143ES70 / 11143ES80
規(guī)格	50 T / 200 T / 500 T

組分信息

組分編號	組分名稱	11143ES50	11143ES70	11143ES80
11143-A	2×Hifair® Ⅲ SG buffer*	500 μL	4×500 μL	5 mL
11143-B	Hifair® UH Ⅲ Enzymes	50 μL	200 μL	500 μL
11143-C	Hieff® 50×High Rox	50 μL	200 μL	500 μL
11143-D	Hieff® 50×Low Rox	50 μL	200 μL	500 μL
11143-E	RNase-free ddH₂O	500 μL	4×500 μL	5 mL

*SG buffer為SYBR Green buffer的簡寫，UH為UNICON-Hifair的簡寫。

儲存條件

-25~-15℃避光儲存，有效期1年。本品避免反復(fù)凍融。建議分裝保存。

使用說明

組分	體積（μL）	終濃度
2×Hifair® Ⅲ SG buffer	10	1×
Hifair® UH Ⅲ Enzymes	1	-
Hieff® 50×High Rox or Low Rox^b	0.4	1×
Forward Primer (10 μmol/L)	0.4	0.2 μmol/L^a
Reverse Primer (10 μmol/L)	0.4	0.2 μmol/L^a
模板 RNA^c	Total RNA 1 pg-1 μg	-
RNase-free ddH₂O	to 20^d	-

表1反應(yīng)體系(以20 μL為例)**

**使用前務(wù)必充分混勻，避免劇烈震蕩產(chǎn)生過多氣泡。

a. 引物濃度：通常引物終濃度為0.2 μmol/L，也可以根據(jù)情況在0.1-1.0 μmol/L之間進(jìn)行調(diào)整。

b. 參比染料：Rox的添加，可根據(jù)不同儀器型號進(jìn)行選擇，具體可參考【適用機(jī)型】。

c. 模板稀釋：qPCR靈敏度極高，建議將模板進(jìn)行稀釋使用，控制CT值在20-30之間適宜。

d. 反應(yīng)體系：推薦使用20 μL或50 μL，以保證目的基因擴(kuò)增的有效性和重復(fù)性。

e. 體系配制：請于超凈工作臺內(nèi)配制，并使用無核酸酶殘留的槍頭、反應(yīng)管；推薦使用帶濾芯的槍頭。避免交叉污染和氣溶膠污染。

循環(huán)步驟	溫度	時間	循環(huán)數(shù)
逆轉(zhuǎn)錄	42℃^b	10 min^a	1
預(yù)變性	95℃	5 min	1
擴(kuò)增反應(yīng)	95℃	10 sec	40
擴(kuò)增反應(yīng)	60℃	30 sec	40
熔解曲線^d	儀器默認(rèn)設(shè)置		1

表2 標(biāo)準(zhǔn)擴(kuò)增程序***

***針對實驗需求可做適當(dāng)調(diào)整。

a. 逆轉(zhuǎn)錄：對于一些極低豐度的基因可以將逆轉(zhuǎn)錄時間適當(dāng)延長至15 min。

b. 溫度：對于含復(fù)雜二級結(jié)構(gòu)或高GC含量的RNA模板，可將逆轉(zhuǎn)錄溫度提高到55℃，有助于提升靈敏度。

c. 熒光信號采集：不同的qPCR檢測儀器所需的熒光信號采集時間不同，請根據(jù)最短時間限制進(jìn)行設(shè)置。

d. 熔解曲線：通常情況下可以使用儀器默認(rèn)程序。

適用機(jī)型

High Rox適用機(jī)型： ABI 5700, 7000, 7300, 7700, 7900HT Fast, StepOne, StepOne Plus;

Low Rox適用機(jī)型： ABI 7500, 7500 Fast, ViiA7, QuantStudio 3 and 5, QuantStudio 6, 7, 12k Flex;

Stratagene MX3000P, MX3005P, MX4000P;

不需要Rox校正的儀器型號：

Bio-Rad: CFX96, CFX384, iCycler iQ, iQ5, MyiQ, MiniOpticon, Opticon, Opticon 2, Chromo4;

Eppendorf: Mastercycler ep realplex, realplex 2 s;

Qiagen: Corbett Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000;

Roche Applied Science: LightCycler 480, LightCycler2.0, Lightcycler 96;

Thermo Scientific: PikoReal Cycler; Cepheid: SmartCycler; Illumina: Eco qPCR.

注意事項

實驗過程中請使用RNase-free耗材。
為了您的安全和健康，請穿實驗服并佩戴一次性手套操作。
本產(chǎn)品僅作科研用途！

Ver.CN20230404

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