IF>5000經(jīng)典預(yù)混液Hieff? qPCR SYBR Green Master Mix(Low Rox Plus)
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產(chǎn)品說(shuō)明書(shū)
FAQ
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產(chǎn)品簡(jiǎn)介
Hieff® qPCR SYBR Green Master Mix(Low Rox Plus)是2×實(shí)時(shí)定量PCR擴(kuò)增的預(yù)混合溶液。Mix中含有熱啟動(dòng)Hieff® DNA Polymerase、SYBR Green I、dNTPs、Mg2+及Low Rox。使用時(shí)，僅需在擴(kuò)增體系中加入模板和引物即可進(jìn)行實(shí)時(shí)熒光定量PCR，大大簡(jiǎn)化操作過(guò)程，降低污染幾率。
本品采用的DNA聚合酶配體可以隨溫度變化實(shí)時(shí)調(diào)節(jié)DNA聚合酶活性。配方添加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，使定量PCR可以在寬廣的定量區(qū)域內(nèi)獲得良好的線性關(guān)系。
產(chǎn)品信息
|
貨號(hào) |
11202ES03 / 11202ES08 / 11202ES50/ 11202ES60 |
|
規(guī)格 |
1 mL / 5×1 mL / 50×1 mL / 100×1 mL |
儲(chǔ)存條件
-25~-15℃避光保存，有效期18個(gè)月。本品避免反復(fù)凍融。產(chǎn)品中含有熒光染料SYBR Green I，保存或配制反應(yīng)體系時(shí)需避免強(qiáng)光照射。
使用說(shuō)明
- 反應(yīng)體系（推薦冰上配制）
|
組分 |
體積 (μL)**** |
體積 (μL) |
終濃度 |
|
Hieff® qPCR SYBR Green Master Mix (Low Rox Plus)* |
25 |
10 |
1× |
|
Forward Primer (10 μM)** |
1 |
0.4 |
0.2 μM |
|
Reverse Primer (10 μM)** |
1 |
0.4 |
0.2 μM |
|
模板DNA*** |
X |
X |
- |
|
RNase Free H2O |
to 50 |
to 20 |
- |
*使用前務(wù)必充分混勻，避免劇烈震蕩產(chǎn)生過(guò)多氣泡。
**通常引物終濃度為0.2 μM，也可以根據(jù)情況在0.1-1.0 μM之間進(jìn)行調(diào)整。
***如模板類(lèi)型為未稀釋cDNA原液，使用體積不應(yīng)超過(guò)qPCR反應(yīng)總體積的1/10。cDNA原液建議5-10倍稀釋?zhuān)罴涯０寮尤肓恳詳U(kuò)增得到的CT值在20-30個(gè)循環(huán)為好。
****推薦使用20 μL或50 μL總體積，以保證目的基因擴(kuò)增的有效性和重復(fù)性。
- 擴(kuò)增程序*
- 兩步法
|
循環(huán)步驟 |
溫度 |
時(shí)間 |
循環(huán)數(shù) |
|
預(yù)變性** |
95℃ |
5 min |
1 |
|
變性 |
95℃ |
10 sec |
40 |
|
退火/延伸*** |
60℃ |
30 sec**** |
|
|
熔解曲線階段***** |
儀器默認(rèn)設(shè)置 |
1 |
|
2）三步法
|
循環(huán)步驟 |
溫度 |
時(shí)間 |
循環(huán)數(shù) |
|
預(yù)變性** |
95℃ |
5 min |
1 |
|
變性 |
95℃ |
10 sec |
40 |
|
退火*** |
55-60℃ |
20 sec |
|
|
延伸 |
72℃ |
20 sec**** |
|
|
熔解曲線階段***** |
儀器默認(rèn)設(shè)置 |
1 |
|
*高特異性可選擇兩步法，高效率擴(kuò)增可選擇三步法。
**預(yù)變性時(shí)間可根據(jù)不同模板和引物的具體情況適當(dāng)縮短至2 min。
***退火溫度和時(shí)間請(qǐng)根據(jù)引物和目的基因的長(zhǎng)度進(jìn)行調(diào)整。
****熒光信號(hào)采集請(qǐng)按照儀器使用說(shuō)明書(shū)要求進(jìn)行實(shí)驗(yàn)程序設(shè)置，幾種常見(jiàn)儀器的時(shí)間設(shè)定如下：
30sec以上：Applied Biosystems: StepOne, StepOne Plus, 7500 Fast；Roche Applied Science: LightCycler 480；Bio-Rad: CFX96
31sec以上：Applied Biosystems: 7300
34sec以上：Applied Biosystems: 7500
*****熔解曲線通常情況下可以使用儀器默認(rèn)程序。
- 結(jié)果分析
定量實(shí)驗(yàn)至少需要三個(gè)生物學(xué)重復(fù)。反應(yīng)結(jié)束后需要確認(rèn)擴(kuò)增曲線及熔解曲線。
1）擴(kuò)增曲線
- Ct值落在20-30之間時(shí)，定量分析最準(zhǔn)確；
- Ct值小于10，需要將稀釋模板后，重新進(jìn)行實(shí)驗(yàn)；
- Ct值介于30-35之間時(shí)，需要提高模板濃度，或增大反應(yīng)體系的體積，以提高擴(kuò)增效率，保證結(jié)果分析的準(zhǔn)確性；
- Ct值大于35時(shí)，檢測(cè)結(jié)果無(wú)法定量分析基因的表達(dá)量，但可用于定性分析。
2）熔解曲線
a. 熔解曲線單峰，表明反應(yīng)特異性好可以進(jìn)行定量結(jié)果分析；若熔解曲線出現(xiàn)雙峰或者多峰，則不能進(jìn)行定量分析。
b. 熔解曲線出現(xiàn)雙峰，需要通過(guò)DNA瓊脂糖凝膠電泳判斷非目標(biāo)峰是引物二聚體還是非特異性擴(kuò)增。
c. 如果是引物二聚體，建議降低引物濃度，或者重新設(shè)計(jì)擴(kuò)增效率高的引物。
d. 如果是非特異性擴(kuò)增，請(qǐng)?zhí)岣咄嘶饻囟?，或者重新設(shè)計(jì)更高特異性的引物。
- 引物設(shè)計(jì)指南
1） 推薦引物長(zhǎng)度25 bp左右。擴(kuò)增產(chǎn)物長(zhǎng)度150 bp為佳，可以在100 bp-300 bp內(nèi)選擇。
2） 正向引物和反向引物的Tm值相差不宜超過(guò)2℃。引物Tm值60℃-65℃為佳。
3) 引物堿基分布要均勻，避免出現(xiàn)連續(xù)的4個(gè)相同堿基，GC含量控制在50%左右。3’端最后一個(gè)堿基最好為G或C。
4) 引物內(nèi)部或者正反兩條引物間最好避免出現(xiàn)有3個(gè)堿基以上的互補(bǔ)序列。
5) 引物特異性需要用NCBI BLAST程序進(jìn)行核對(duì)。避免引物3’端有2個(gè)堿基以上的非特異性互補(bǔ)。
6) 設(shè)計(jì)完成的引物需要進(jìn)行擴(kuò)增效率的檢測(cè)，只有具備相同擴(kuò)增效率的引物才可用于定量比較分析。
5. 適用機(jī)型
Applied Biosystems: 7500, 7500 Fast, ViiA7, QuantStudio Dx, QuantStudio 3 and 5, QuantStudio 6,7,12k Flex;
Stratagene: MX3000P, MX3005P, MX4000.
注意事項(xiàng)
- 推薦使用本公司cDNA合成試劑盒（Cat#11141），以有效去除RNA樣品中殘留的基因組。
- 解凍后Master Mix可能出現(xiàn)絮狀物質(zhì)，4℃放置并上下顛倒混勻至溶液澄清，不影響試劑性能。
- 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。
- 本產(chǎn)品僅作科研用途。
Ver.CN20231128
Q：建議qPCR 實(shí)驗(yàn)用幾步法？
A：常用 2 步法。需提高擴(kuò)增特異性，可選用 2 步法或提高退火溫度。在擴(kuò)增效率低， ct 值過(guò)大的時(shí)候，可以改用 3 步法或延長(zhǎng)延伸時(shí)間。
Q：預(yù)變性 5 min 調(diào)整成了 10min，對(duì)于實(shí)驗(yàn)結(jié)果有影響嗎？
A：有影響，預(yù)變性時(shí)間較長(zhǎng)可能會(huì)影響酶的活性，導(dǎo)致 PCR 產(chǎn)物的產(chǎn)量有所降低。Q：qPCR 實(shí)驗(yàn)結(jié)果的有效性？為什么建議Ct 值要大于 15？
A：有效性要滿(mǎn)足三個(gè)條件：（1）標(biāo)準(zhǔn)曲線：擴(kuò)增效率范圍:90-110%，對(duì)應(yīng)斜率為 -3--3.5。 R2＞0.98。 (擴(kuò)增效率=10-1/斜率-1)，當(dāng)斜率=-3.32 時(shí)，擴(kuò)增效率=100%。（2）擴(kuò) 增曲線：S 型曲線，且 Ct 值在 15－35 之間，陰性對(duì)照 Ct＞35 或無(wú) Ct 值。（3）熔解曲線：為單一峰。
Ct 值大于 15 個(gè)循環(huán)是因?yàn)?/font> 3-15 個(gè)循環(huán)的熒光值標(biāo)準(zhǔn)差的 10 倍是熒光閾值，Ct 值太小了會(huì)影響曲線。
Q：同一基因復(fù)孔間熔解曲線 Tm 值有差異？
A：同樣的擴(kuò)增產(chǎn)物也會(huì)出現(xiàn)Tm 值有微小差異，一般差異在 1 度以?xún)?nèi)都可以接受。
Q：為什么稀釋了模板CT 值反而變小了？
A:一般 CT 值與模板起始濃度呈負(fù)相關(guān)，濃度越高，CT 值越小。但也有很多特殊情況， 比如體系中存在抑制物或是模板不純，這時(shí)候稀釋模板反而能使 CT 值變低。
Q：內(nèi)參CT 值小于 20，目的基因均大于 30，怎么辦？
A：可能是目的基因?yàn)榈拓S度表達(dá)基因?qū)е?。建議：a）換用內(nèi)參；b）換引物；c）換檢測(cè)線性范圍更廣的qPCR mix。
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