Hifair? Ⅱ 1st Strand cDNA Synthesis Kit
批量采購(gòu)， 請(qǐng)點(diǎn)擊詢價(jià)
產(chǎn)品說(shuō)明書(shū)
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產(chǎn)品描述
Hifair® Ⅱ 1st Strand cDNA Synthesis Kit是基于Hifair® Ⅱ Reverse Transcriptase開(kāi)發(fā)的試劑盒。與Hieff® M-MLV (H-) Reverse Transcriptase相比，Hifair® Ⅱ Reverse Transcriptase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。Hifair® Ⅱ Reverse Transcriptase合成全長(zhǎng)cDNA的能力也有了提升，可擴(kuò)增長(zhǎng)達(dá)10 kb的cDNA。
試劑盒中包含由總RNA或mRNA合成高質(zhì)量第一鏈cDNA所需的所有成分，并提供兩種cDNA合成引物：Random Primers N6和oligo (dT)18，合成的單鏈cDNA產(chǎn)物可直接用來(lái)進(jìn)行后續(xù)PCR或者qPCR反應(yīng)。
產(chǎn)品組分
|
編號(hào) |
組分 |
產(chǎn)品編號(hào)/規(guī)格 |
|
11119ES60 (100 T) |
||
|
11119-A |
RNase-free H2O |
2×1 mL |
|
11119-B |
5×Hifair® Ⅱ Buffer |
400 μL |
|
11119-C |
Hifair® Ⅱ Enzyme Mix |
200 μL |
|
11119-D |
Oligo (dT)18 (50 μM) |
100 μL |
|
11119-E |
Random Primers N6 (50 μM) |
100 μL |
【注】：1）5×Hifair® Ⅱ Buffer中包含dNTP。2）Hifair® Ⅱ Enzyme Mix中包含RNase inhibitor。
運(yùn)輸和保存方法
冰袋運(yùn)輸。-20℃保存。
注意事項(xiàng)
1）反應(yīng)液的配制應(yīng)在冰上操作完成，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！
第一鏈cDNA合成操作步驟
1. 逆轉(zhuǎn)錄反應(yīng)體系配制（20 μL體系）
|
組分 |
使用量 |
|
RNase-free H2O |
To 20 μL |
|
Total RNA |
1 ng -5 μg* |
|
or mRNA |
1 ng-500 ng* |
|
5×Hifair® Ⅱ Buffer |
4 μL |
|
Random Primers N6 (50 μM) |
1 μL |
|
or Oligo (dT)18 (50 μM) |
or 1 μL |
|
or Gene Specific Primers (2 μM) |
or 1 μL |
|
Hifair® Ⅱ Enzyme Mix |
2 μL |
【可省略】：若后續(xù)實(shí)驗(yàn)為PCR，針對(duì)復(fù)雜模板，可將RNA、H2O、反轉(zhuǎn)錄引物在65℃孵育5 min 后，迅速冰上冷卻，然后再加入Hifair® Ⅱ Enzyme Mix；若后續(xù)實(shí)驗(yàn)為qPCR，則可省略65℃孵育步驟，直接在體系中加入Hifair® Ⅱ Enzyme Mix。
【注】：* 若后續(xù)實(shí)驗(yàn)為qPCR，建議Total RNA 或 mRNA投入量不超過(guò)1 μg或100 ng，如果目的基因的表達(dá)豐度非常低，最多可投入5 μg Total RNA或500 ng mRNA。
2. 逆轉(zhuǎn)錄程序設(shè)置
|
溫度 |
時(shí)間 |
|
25℃ |
5 min |
|
42℃ |
30 min |
|
85℃ |
5 min |
【注】：
1）熒光定量實(shí)驗(yàn)可以只使用Random Primers N6；也可以1:1與Oligo (dT)18混合使用，效果更好。
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℃。
3）逆轉(zhuǎn)錄產(chǎn)物可以-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。
關(guān)于引物的選擇
1） 如果模板為真核生物來(lái)源，建議選擇Oligo (dT)18 ，與真核生物mRNA的3’ Poly A尾配對(duì)，可獲得最高產(chǎn)量的全長(zhǎng)cDNA。
2）原核生物RNA的反轉(zhuǎn)錄請(qǐng)選用Random Primers N6或者基因特異性引物。
3）Random Primers N6適用性較廣，mRNA、rRNA、tRNA、small RNA和LncRNA等模板均可用Random Primers N6進(jìn)行反轉(zhuǎn)錄。
4）使用Random primers N6，進(jìn)行2 kb以下的cDNA合成時(shí)，Random primers N6的使用量為 1-2 μL；2 kb 以上的cDNA合成時(shí)，Random primers N6 的使用量為0.4-1 μL。
相關(guān)產(chǎn)品
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貨號(hào) |
規(guī)格 |
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11119ES60 |
100 T |
|
|
11120ES60 |
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|
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11202ES08 |
5 mL |
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11203ES08 |
5 mL |
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Hieff UNICON® Power qPCR SYBR Green Master Mix ( 抗體法，No Rox) |
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5 mL |
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Hieff UNICON® Power qPCR SYBR Green Master Mix ( 抗體法，Low Rox) |
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5 mL |
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Hieff UNICON® Power qPCR SYBR Green Master Mix ( 抗體法，High Rox) |
11197ES08 |
5 mL |
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11198ES08 |
5 mL |
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11199ES08 |
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11200ES08 |
5 mL |
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11184ES08 |
5 mL |
HB210719
Q：A2800，promage 的貨號(hào)， 用我們哪款反轉(zhuǎn)錄替換比較好點(diǎn)？
A：11119 是兩種引物分開(kāi)的，可以做 oligo（dT）和隨機(jī)引物分開(kāi)的，也可以混合的。如果客戶只做定量的話，可以推 11137
Q：?jiǎn)栆幌?11110 這款反轉(zhuǎn)錄單酶，這款產(chǎn)品里頭的組分有沒(méi)有那款kit 或者 mix 也包含呢？
A：11120ES、11123ES、11119ES、11121ES
Q：Hifair Ⅱ 1st Strand cDNA Synthesis Kit 這個(gè)跟諾唯贊的 HiScript II Reverse Transcriptase 區(qū)別？
A：如果客戶不要求逆轉(zhuǎn)溫度達(dá)到 55 度，是可以的，我們?nèi)孓D(zhuǎn)錄酶優(yōu)于這個(gè)。
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