Hifair? Ⅱ 1st Strand cDNA Synthesis SuperMix
批量采購(gòu)， 請(qǐng)點(diǎn)擊詢價(jià)
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產(chǎn)品描述
Hifair® Ⅱ 1st Strand cDNA Synthesis SuperMix為即用型預(yù)混液，包含Hifair® Ⅱ Reverse Transcriptase，RNase Inhibitor，dNTP，Random primers/Oligo dT Primer mix和優(yōu)化的緩沖體系，只需再加入模板RNA和RNase-free H2O即可進(jìn)行反應(yīng)。Hifair® Ⅱ Reverse Transcriptase是在Hieff® M-MLV (RNase H-) Reverse Transcriptase基礎(chǔ)上通過(guò)基因工程技術(shù)得到新的逆轉(zhuǎn)錄酶，與Hieff® M-MLV (RNase H-) Reverse Transcriptase相比，其熱穩(wěn)定性大幅度提高，可耐受高達(dá)50℃的反應(yīng)溫度，適合具有復(fù)雜二級(jí)結(jié)構(gòu)的RNA模板的逆轉(zhuǎn)錄。同時(shí)，該酶增強(qiáng)了與模板的親和力，適合少量模板以及低拷貝基因的逆轉(zhuǎn)錄。
產(chǎn)品組分
|
編號(hào) |
組分 |
產(chǎn)品編號(hào)/規(guī)格11120ES60 (100 T) |
|
11120-A |
RNase-free H2O |
2×1 mL |
|
11120-B |
2×Hifair® Ⅱ SuperMix |
1 mL |
產(chǎn)品應(yīng)用
適用于RT-qPCR實(shí)驗(yàn)。
運(yùn)輸和保存方法
冰袋運(yùn)輸。-20℃保存，有效期18個(gè)月。
第一鏈cDNA合成操作步驟
|
逆轉(zhuǎn)錄反應(yīng)體系 |
逆轉(zhuǎn)錄程序 |
||
|
組分 |
使用量 |
溫度 |
時(shí)間 |
|
RNase free H2O |
To 20 μL |
25℃ |
5 min |
|
2×Hifair® Ⅱ SuperMix |
10 μL |
42℃ |
30 min |
|
Total RNA |
1 ng -5 μg |
85℃ |
5 min |
|
or mRNA |
1 ng-500 ng |
||
【注】：
1. 20 μL逆轉(zhuǎn)錄反應(yīng)體系建議Total RNA的投入量不超過(guò)1 μg。如果目的基因的表達(dá)豐度低，最多投入5 μg Total RNA；
2. 逆轉(zhuǎn)錄溫度：推薦使用42℃。對(duì)于高GC含量模板或者復(fù)雜模板，可將逆轉(zhuǎn)錄溫度提高到50℃。
※ 逆轉(zhuǎn)錄產(chǎn)物可立即用于后續(xù)qPCR反應(yīng)，也可-20℃短期保存，若需長(zhǎng)期保存，建議分裝后，于-80℃保存，避免反復(fù)凍融。
注意事項(xiàng)
1）所有操作均應(yīng)在冰上進(jìn)行，且操作過(guò)程應(yīng)避免RNase污染；
2）為了您的安全和健康，請(qǐng)穿實(shí)驗(yàn)服并佩戴一次性手套操作。
3）本產(chǎn)品僅作科研用途！
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HB210719
Q：逆轉(zhuǎn)錄反應(yīng)中的引物如何選擇？
A：根據(jù)實(shí)驗(yàn)?zāi)康牟煌?，可按下列建議選擇（選擇指南可見下表）：
a）對(duì)于全長(zhǎng)第一鏈 cDNA 合成，且模板為真核生物來(lái)源，推薦選擇Oligo (dT)引物。
b）當(dāng)目標(biāo)區(qū)域具有復(fù)雜二級(jí)結(jié)構(gòu)或模板為原核生物來(lái)源，推薦選擇 Random Primers 引物。
c）基因特異性引物（GSP）是與模板序列互補(bǔ)的引物，適用于目的序列已知的情況。
d）若逆轉(zhuǎn)錄后續(xù)為 qPCR 實(shí)驗(yàn)，可將 Oligo (dT)與Random Primers 混合使用。
|
逆轉(zhuǎn)錄引物選擇指南 |
||||
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|
特征 |
優(yōu)點(diǎn) |
缺點(diǎn) |
結(jié)合方式 |
|
Oligo (dT) |
1）12-20 個(gè)T； 2）與真核生物 mRNA 的3 ’ Poly A 尾配對(duì)。 |
可 合 成 全長(zhǎng) cDNA。 |
1 ） 僅擴(kuò)增有 polyA尾 的mRNA； 2 ） 對(duì)模板質(zhì) 量 要 求高。 |
|
|
Random Primers |
1）6-9 個(gè)堿基； 2）可隨機(jī)識(shí)別模板并結(jié)合 |
適 合 復(fù) 雜結(jié) 構(gòu) 和 微量模板。 |
特異性低， 小片段多。 |
|
|
基因特異性性引物 （GSP） |
識(shí)別特定模板序列。 |
特異性強(qiáng)， 靈敏度高。 |
僅 合 成 特定的序列。 |
|
A：有 A 尾的 lncRNA 用預(yù)混液和 KIT （ 11123/11141/11121/11139） 都可以， circRNA、miRNA 和無(wú) A 尾的 lncRNA 用 KIT（11121/11139）。microRNA 需要特殊的莖環(huán)引物，需特別針對(duì)的逆轉(zhuǎn)錄試劑盒。
Q：能否用來(lái)做 miRNA/circRNA/lncRNA 的逆轉(zhuǎn)錄？
Q：克隆逆轉(zhuǎn)和定量逆轉(zhuǎn)有什么區(qū)別？克隆逆轉(zhuǎn)產(chǎn)物和定量逆轉(zhuǎn)產(chǎn)物可以相互嗎？
A：a）目的不同：克隆逆轉(zhuǎn)后的 cDNA 后續(xù)用于基因克隆，后續(xù)實(shí)驗(yàn)以普通 PCR 為主； 定量逆轉(zhuǎn)后的 cDNA 后續(xù)用于基因定量，后續(xù)實(shí)驗(yàn)以qPCR 為主。
b）逆轉(zhuǎn)錄過(guò)程不同：克隆逆轉(zhuǎn)使用 Oligo dT，保證合成的長(zhǎng)度。隨機(jī)引物使用較少； 定量逆轉(zhuǎn)使用 Oligo dT 或隨機(jī)引物，或兩者混合使用。
c）克隆逆轉(zhuǎn)產(chǎn)物可用于 qPCR，但定量逆轉(zhuǎn)產(chǎn)物不推薦用于普通 PCR，定量逆轉(zhuǎn)試劑中含 Oligo (dT)與 Random Primers 混合引物，產(chǎn)物長(zhǎng)度較短，可做短片段 PCR，過(guò)長(zhǎng)的不適合。
Q：逆轉(zhuǎn)錄試劑盒可以逆轉(zhuǎn)真菌（或其他物種）RNA 嗎？有沒(méi)有專門針對(duì)真菌（或其他物種）的RNA 逆轉(zhuǎn)錄的試劑盒呢？
A：RNA 的物種與逆轉(zhuǎn)錄沒(méi)有很大關(guān)系，RNA 質(zhì)量與逆轉(zhuǎn)錄有關(guān)。所以，得到 RNA 后， 逆轉(zhuǎn)錄試劑盒都可以用，沒(méi)有特別針對(duì)的。
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