Polymerase Chain Reaction is a method of replicating DNA exponentially from even a single molecule.

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Real time PCR normalization algorithm

When performing normalization of real time PCR results, I found two ways of doing it: In my lab they follow the next layout: $\text{Efficiency}^{-(CT\ _{\large\text{interest gene}} - CT ...
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573 views

Real time PCR standard curve

As blunt as possible: when performing real time PCR it is a routine step to run one PCR in order to plot a "standard curve" with several decreasing dilution ratios from your sample. what is the real ...
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377 views

How are DNA segments selected in PCR?

I understand that in PCR we're able to amplify only selected portions of the DNA... however despite reading it from multiple sources, I cannot figure out how this selection actually takes place. I ...
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1answer
558 views

Effect of single nucleotide deletion or insertion on primer annealing

How is primer annealing, and, consequently, PCR amplification affected by single nucleotide deletion or insertion inside the primer ? Imagine a primer like this: GCGTCATAAAGGGGACGTG (primer) and ...
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747 views

Are there issues with filling PCR tubes to capacity?

I'm planning to scale up a PCR reaction, and I'm wondering if filling the PCR tubes to the maximum volume of 200 ul would be a problem. It would mean a lot less pipetting as I would only need 1/4 of ...
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2answers
9k views

Does an annealing temp higher than primer's Tm contribute to primer dimer?

I am attempting to reproduce results from a number of journal articles all referring to the same SNP. In doing this I'm using the same primer set outlined in the articles. When I attempted a run the ...
5
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131 views

Sequencing from PCR

As far as I understand it, PCR can be used to make many copies of one gene. My question is, is it possible to sequence DNA after PCR and is it easier than sequencing it via other methods. If it is ...
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1answer
87 views

Correct/complete representation of RTPCR statistics

In papers reporting a relative quantification of gene expression by RTPCR, I often see a bar chart with mean ± standard error or deviation, with the deviation belonging to biological replicates. This ...
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0answers
32 views

What evidence is there that common PCR DNA purification kits can isolate DNA from pathogens engulfed in macrophages?

I have previously asked a more general question about how PCR sequencing reaches all possible DNA targets here. I want to ask more specifically now: what evidence is there that common PCR DNA ...
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Does common PCR amplify genes regardless of what cells / barriers they are in?

I have some understanding of how PCR testing works. What I have always been wondering: how can we be sure that a primer reacts with the targeted gene(s) regardless of where¹ the genes are inside a ...
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1answer
42 views

Why do you need primers in PCR? [duplicate]

I have read that DNA polymerase requires a primer to bind to the DNA, but I am confused as to why this is the case. When DNA undergoes replication in the cell, there are no primers in the nucleus so ...