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★彥琳★ asked in 科學化學 · 1 decade ago

PT-PCR ~~急!

2.5. Quantification of living S. mutans (real-time


2.5.1. RNA extraction and cDNA synthesis

The plates were washed with phosphate-buffered saline

(PBS) solution (pH 7.2) to remove unattached cells, and total

RNA was extracted from the remaining S. mutans attached

onto HAp and the resin coatings using Trizol LS Reagent

(Invitrogen, Life Technologies, Carlsbad, CA, USA) according

to the manufacturer’s instructions. Contaminated genomic

DNA was removed by DNase I (Takara Bio, Shiga, Japan)

with RNase Inhibitor (Invitrogen, Life Technologies, Carlsbad,

CA, USA). First-strand cDNA synthesis was performed using

SuperScriptTM II RT (Invitrogen, Life Technologies, Carlsbad,

CA, USA) with random primers in accordance with the manufacturer’s


2.6. Real-time RT-PCR

Living S. mutans was quantified by real-time RT-PCR.

GeneAmpR 5700 Sequence Detection System (PE Applied

Biosystems, Foster City, CA, USA) was used for monitoring the

fluorescence from dsDNA-binding SYBR Green I. Quantitative

PCR was performed using universal primers for bacterial 16S

rRNA gene, as described previously [16]. Briefly, the PCR mixture

was prepared to contain 2× SYBR Green PCR Master Mix

(PE Applied Biosystems, Foster City, CA, USA), 20 pmol of forward

and reverse primer and 2.5l of synthesized cDNA. The

thermo-cycling program was 40 cycles of 95 ◦C for 15 s and

60 ◦C for 1min with an initial cycle of 95 ◦C for 10 min. A dissociation

curve (melting curve) was constructed in the range

of 60–95 ◦C, and the data were analyzed using the GeneAmp

5700 SDS software. Duplicate measurements were performed

for each sample.




1 Answer

  • VBC
    Lv 7
    1 decade ago
    Favorite Answer

    2.5 活鏈球菌定量 (使用 即時定量反轉錄PCR 反應)

    2.5.1 RNA萃取與cDNA合成

    培養皿以pH7.2的PBS溶液清洗, 除去未附著的無關細菌. 以Trizol LS Reagent萃取其餘附著於培養皿基質上的鏈球菌, 步驟依產品所附. 萃取後混雜的染色體DNA以DNase I去除, 同時加入RNase Inhibitor. 第一股的 cDNA以SuperScript II RT及random primers(隨機引子)進行反轉錄, 步驟依產品所附.

    2.6 即時定量反轉錄PCR

    以即時定量反轉錄PCR定量存活的鏈球菌. 以GeneAmpR 5700 Sequence Detection System偵測雙股DNA的SYBR Green I螢光訊號. qPCR使用共通的 16S rRNA引子(序列如先前所附, 參見附件16). 快速的將PCR反應物與兩倍濃縮的SYBR Green PCR Master Mix試劑混合. 加入各20 pmol的順向與反向引子及 2.5 μl的cDNA(μ原文應遺漏μ). 溫度循環程式設定為最初先進行95 ◦C, 10分鐘, 然後是 95 ◦C, 15秒 及60 ◦C, 1分鐘 進行 40個循環. 游離曲線(melting curve)執行範圍為60 ~ 95 ◦C. 最後結果以GeneAmp 5700 SDS 軟體進行分析. 每個檢體執行二重複反應.

    95 ◦C, 10分鐘

    95 ◦C, 15秒

    60 ◦C, 1分鐘

    plate read

    Goto step 2 共40次

    melting curve 60 ~ 95 ◦C


    部分實驗內容依照較易了解的中文文法翻譯(特別是PCR溫度設定), 各試劑及儀器廠牌標示省略.

    如需各步驟詳細說明(各產品說明書或實驗原理及建議)或是需要購買本實驗所需的試劑與相關儀器, 敬請與我聯絡

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