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產品信息
產品名稱 | 產品編號 | 規格 | 價格（元） | 促銷價（元） |
Hifair? Ⅱ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) | 11123ES10 | 10 T | 236.00 | / |
Hifair? Ⅱ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) | 11123ES60 | 100 T | 955.00 | 764.00 |
產品描述
Hifair? Ⅱ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus)基于Hifair? Ⅱ Reverse Transcriptase而開發的即用型預混液。該酶的熱穩定性大幅度提高，可耐受高達50℃的反應溫度，適合具有復雜二級結構的RNA模板的逆轉錄。同時，該酶增強了與模板的親和力，適合少量模板以及低拷貝基因的逆轉錄。
該預混液包含gDNA digester和2×Hifair? Ⅱ SuperMix plus。gDNA digester可去除RNA模板中殘留的基因組DNA污染，保證后續結果更加可靠。2×Hifair? Ⅱ SuperMix plus含有逆轉錄反應所需的所有組分（Buffer，dNTP，Hifair? Ⅱ Reverse Transcriptase，RNase inhibitor，Random primers/ Oligo (dT)18 primer mix），只需加入RNA模板和 RNase-free ddH2O即可進行逆轉錄反應，并同時終止gDNA digester的作用，保證cDNA的完整性。
該產品適用于兩步法RT-qPCR檢測，針對qPCR進行Random primers/ Oligo (dT)18 primer的比例優化，使cDNA合成可從RNA轉錄本的各個區域起始，并具有相同的逆轉錄效率，最大程度保證了qPCR結果的真實性和可重復性。逆轉錄產物兼容SYBR? Green和探針法qPCR，可以根據實驗目的，選擇UNICON? qPCR SYBR? Green Master Mix，Hieff? qPCR SYBR? Green Master Mix或Hieff? qPCR TaqMan Probe Master Mix等試劑配合使用，進行高性能的基因表達分析。
產品組分
編號 | 組分 | 產品編號/規格 | |
11123ES10 (10 T) | 11123ES60 (100 T) | ||
11123-A | RNase-free ddH2O | 1 mL | 2×1 mL |
11123-B | 5×gDNA digester Buffer | 20 μL | 200 μL |
11123-C | gDNA digester | 10 μL | 100 μL |
11123-D | 2×Hifair? Ⅱ SuperMix plus | 100 μL | 1 mL |
【注】：2×Hifair? Ⅱ SuperMix plus含有gDNA digester抑制劑。
運輸和保存方法
干冰運輸。-20℃保存。有效期18個月。
注意事項
1）gDNA digester和2×Hifair? Ⅱ SuperMix plus含有高濃度的甘油，使用前請短暫離心，吹打混勻。
2）建議RNA是溶于水而不是TE Buffer中，因為TE Buffe會干擾gDNA去除以及逆轉錄反應。
3）可以不經過基因組去除步驟，直接進行逆轉錄，這樣所得到的結果與使用Hifair? Ⅱ 1st Strand cDNA Synthesis SuperMix (Cat No. 11120ES) 效果一致。但是請勿將gDNA digester與11120ES中的2×Hifair? Ⅱ SuperMix配套使用，因其不含終止gDNA digester反應的成分，會影響反轉錄和后續的qPCR實驗。
4）反應液的配制應在冰上操作完成，操作過程應避免RNase污染。
5) 為了您的安全和健康，請穿實驗服并佩戴一次性手套操作。
6) 本產品僅作科研用途！
第一鏈cDNA合成操作步驟
1. 殘留基因組DNA去除
在RNase free離心管中配制如下混合液，用移液器輕輕吹打混勻。42℃ 孵育2 min。
組分 | 使用量 |
RNase free ddH2O | To 10 μL |
5×gDNA digester Buffer | 2 μL |
gDNA digester | 1 μL |
Total RNA | 1 ng -5 μg* |
or mRNA | 1 ng-500 ng* |
【注】：* 20 μL逆轉錄反應體系建議Total RNA的投入量不超過1 μg。如果目的基因的表達豐度低，最多投入5 μg Total RNA，否則RNA投入量過高，可能會超過后續定量PCR的線性范圍。
2. 逆轉錄反應體系配制（20 μL體系）
在第1步的反應管中直接加入2×HifairTM Ⅱ SuperMix plus，用移液器輕輕吹打混勻。
組分 | 使用量 |
第1步的反應液 | 10 μL |
2×Hifair? Ⅱ SuperMix plus | 10 μL |
3. 逆轉錄程序設置
標準程序（適用于微量和常規量的模板量）
溫度 | 時間 |
25℃ | 5 min |
42℃ | 30 min |
85℃ | 5 min |
【注】：逆轉錄溫度：推薦使用42℃。對于高GC含量模板或者復雜模板，可將逆轉錄溫度提高到50℃。
4. 逆轉錄產物可立即用于qPCR反應，也可-20℃短期保存，若需長期保存，建議分裝后，于-80℃保存，避免反復凍融。
相關產品
產品名稱 | 貨號 | 規格 |
11119ES60 | 100 T | |
11120ES60 | 100 T | |
Hifair? II 1st Strand cDNA Synthesis Kit (gDNA digester plus) | 11121ES60 | 100 T |
Hifair? Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) | 11141ES60 | 100 T |
11201ES08 | 5 mL | |
11202ES08 | 5 mL | |
11203ES08 | 5 mL | |
Hieff UNICON? Power qPCR SYBR Green Master Mix ( 抗體法，No Rox) | 11195ES08 | 5 mL |
Hieff UNICON? Power qPCR SYBR Green Master Mix ( 抗體法，Low Rox) | 11196ES08 | 5 mL |
Hieff UNICON? Power qPCR SYBR Green Master Mix ( 抗體法，High Rox) | 11197ES08 | 5 mL |
11198ES08 | 5 mL | |
11199ES08 | 5 mL | |
11200ES08 | 5 mL | |
11184ES08 | 5 mL |
HB210628
Q：逆轉錄產品如何選擇？
A：參考下表。
產品名稱/貨號 | 產品形式 | 引物 | 適用實驗類型 | 產品特點/適用范圍（A/B） |
Hifair? II Reverse Transcriptase 第二代耐熱逆轉錄酶 11110ES | 單酶 | 無 | ?cDNA產物后續用于PCR ?cDNA產物后續用于qPCR | A．常規表達豐度RNA逆轉錄（總RNA 1ng-5μg） |
Hifair? III Reverse Transcriptase 第三代耐熱逆轉錄酶 11111ES | B.低表達豐度RNA逆轉錄 （總RNA 10pg-5μg）；復雜RNA | |||
Hifair? II 1st Strand cDNA Synthesis Kit 11119ES/11121ES | 酶和Buffer分開 | 單獨成管的Oligo dT 、Random primer | 同A | |
Hifair? III 1st Strand cDNA Synthesis Kit 11135ES/11139ES | 同B | |||
Hifair? II 1st Strand cDNA Synthesis SuperMix for qPCR 11120ES/11123ES | 酶和Buffer預混 | 按比例預混的Oligo dT 、Random primer | ?cDNA產物后續用于qPCR | 同A |
Hifair? III 1st Strand cDNA Synthesis SuperMix for qPCR 11137ES/11141ES | 同B |
Q：逆轉錄引物如何選擇？
A：11123ES中使用的是Random Primers和oligo dT按照一定優化的比例預混引物。
Q：反轉錄產物可以做普通PCR嗎？
A：不推薦。11123ES以預混液形式提供，預混液中包含Oligo (dT)與Random Primers混合引物，逆轉錄產物反轉均一，但長度較短，可以做短片段，過長的不適合。若反轉錄產物后續進行PCR，建議用翌圣11121ES，引物選oligo dT。
Q：逆轉錄產物后續進行qPCR，需要稀釋嗎？
A：建議稀釋5-10倍。目的有二：一、提高稀釋倍數，降低移液誤差，提高實驗重復性；二、降低逆轉錄體系中高濃度某些成分對下游qPCR反應的抑制作用。
Q：該產品可以搭配其他品牌的定量試劑使用嗎？
A：可以。逆轉錄產物進行稀釋后，反轉錄相關組分對后續不同品牌的qPCR試劑影響非常小。
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