NTR366L_week9_lecture_RealPCR

NTR366L_week9_lecture_RealPCR - Todays topics Real-time PCR...

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1 Today’s topics Real-time PCR
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2 Real-time PCR It’s a PCR reaction that amplified DNA is simultaneously detected in real time Measure amount of PCR product present at the end of each cycle Highest precision during the exponential phase
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3 Plateau phase Linear phase Exponential phase End point analysis on agarose gels Cycles PCR Product Polymerase Chain Reaction End-point PCR Yes/No; is it there?
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4 Plateau phase Linear phase Exponential phase Cycles PCR Product Polymerase Chain Reaction Quantitative PCR (real-time PCR) How much is there?
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5 Quantitative PCR (qPCR) Questions: Absolute quantitation: How many copies were initially in the sample? Comparative quantitation: How much more (or less) was initially in sample 1 compared to sample 2?
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6 The more template in the original sample, the earlier the reaction reaches the threshold (exponential phase).
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7 Baseline Threshold Ct value Cycles Cycle Threshold (Ct) Value The more template in the original sample, the smaller the Ct value.
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Threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background. The threshold line is set in the exponential phase of the amplification for the most accurate reading. The cycle at which the sample reaches this level is called the Cycle Threshold, Ct . 8
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NTR366L_week9_lecture_RealPCR - Todays topics Real-time PCR...

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