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12 votes

Are there issues with filling PCR tubes to capacity?

(Also too long for a comment.) This question reminded me about an anecdote told by Arthur Kornberg concerning the approach to scaling up that was adopted by Reiji Okazaki (discoverer of Okazaki ...
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12 votes

Are there issues with filling PCR tubes to capacity?

Too long for a comment: I would say it depends on the PCR. I have personally used maximum volumes of 50ul, if I needed to prepare higher volumes, I made one mix which subsequently got distributed to ...
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11 votes

Good pipetting technique?

Here's some step-by-step advice on good pipetting technique: Make sure the pipette is set for the correct volume Ensure the tip is firmly attached Keep the pipette vertical when pipetting Slowly and ...
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10 votes

What is the half-life of dNTPs at 95 °C?

Instead of searching for the stability of dNTPs, consider the stability of their constituent parts. Caveat: ignoring the bonds between the different parts of dNTPs likely misrepresents the stability ...
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9 votes

How does PCR mutagenesis add restriction site near the gene of interest?

This diagram would be easier for you to conceptualize if you instead used the actual nucleotides in the restriction sites and green GGAGAA region along with your gene of interest (GOI). I recommend ...
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7 votes
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Basic question about multiplex PCR

I haven't done this exact experiment. I am just deducing from the known facts about PCR. The product of PF and PR2 will serve ...
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7 votes
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How are DNA segments selected in PCR?

Note: In your PCR program you always set extension time. Case: Product length = 500bp PCR extension time = 50sec Assuming that polymerase adds 1000 nt/min Cycle 1: Strand that binds FP: extends ~...
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7 votes

Is Melanin a PCR Inhibitor?

Melanin is a potent inhibitor of PCR - when you use B16 cells (or any other cell line that produces melanin) you have to purify your sample from it. Unfortunately a simple Phenol-Chloroform extraction ...
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7 votes

Is PCR a DNA cloning technique?

Short answer All of your sources are correct as they are not mutually exclusive. PCR is used to isolate and amplify DNA to yield small quantities of pure target product. Gene cloning can subsequently ...
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6 votes
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Equation for accurate prediction of PCR yield

An expected efficiency for a typical PCR is 80%, meaning each cycle multiplies the copy number of the targeted DNA sequence 1.58 times. Firstly, it makes more sense to refer to the amount of DNA in a ...
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6 votes
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Why do we need to amplify DNA sequences?

This is a question I also remember wondering about when I was younger in school. Now as a professional it's way too obvious to even explain. But i think it's an important and common question, which ...
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5 votes

Real-time PCR result interpretation

You should try another housekeeping gene like GAPDH since the very definition of these genes is that they shouldn't change with your treatment
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5 votes

Possible reasons for DNA getting stuck in well

There is one simple reason for that, your agarose gel is most likely too dense. Depending in the type of agarose, I would prepare a 0.5-0.6% gel at maximum. Synbio gives this list for "standard" ...
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5 votes
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DNA polymerase in PCR (polymerase chain reaction)

No, this does not happen, as the DNA polymerases used for PCR are DNA dependent. This means that they only synthesize DNA when it is bound to DNA. Even if your primers bind to the RNA, the polymerase ...
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5 votes
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Walk me through microsatellite markers and PCR

It's actually very simple. You misunderstood the term polymorph in this situation. A microsatellite is a sequence composed of a short repeated DNA motif. A polymorph satellite is then simply a ...
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5 votes
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Does common PCR amplify genes regardless of what cells / barriers they are in?

There are many methods of DNA isolation, so it is difficult to make broad statements about all situations. Typically, though, during the DNA isolation protocol, essentially all proteins are denatured ...
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5 votes
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Alternatives to PCR

Edited after clarifications in question, Let's start with normal functions of both enzymes. Helicases separates DNA strands while polymerase synthesize DNA strands as shown in the following figure. (...
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5 votes

Is Melanin a PCR Inhibitor?

I don't have much expertise in PCR (so please do not ask me technicalities). A research has proven that melanin is a PCR inhibitor as it forms complexes with thermostable DNA polymerase and inhibits ...
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5 votes
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Why is only one primer used for DNA sequencing instead of two?

The reaction for the Sanger sequencing reacting contains beneath the normal dNTP (which are needed to make most of the DNA) the dideoxynucleotides, which lack the 3'-OH-Group and which are labelled ...
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5 votes

Can primers for PCR be duplicated?

Short answer: You need to buy some more, but you need the sequence also for ordering. Long answer: The Taq polymerase needs a piece of DNA (or RNA) to prime the reaction and be able to enlarge the ...
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5 votes

Why PCR primers don't amplify beyond the target region?

How does DNA synthesis from the forward primer stop when it reaches the point where the reverse primer is? Briefly, it doesn't. You get a lot of single-stranded long pieces of DNA (step 1 here, ...
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Do the primers used for Covid-19 test work with all new strains?

I TRY to answer. First of all, no we cannot be 100% sure that the primers are effective against all strains. However, the strains that are under media attention right now are due to variants in the ...
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5 votes

Is it possible to use PCR to test for Machado-Joseph disease?

This paper describes an allele-specific PCR protocol to detect CAG repeat expansion in MJD patients: Maciel P, Costa MDC, Ferro A, et al. Improvement in the Molecular Diagnosis of Machado-Joseph ...
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4 votes

How are DNA segments selected in PCR?

I'm not completely clear when you say "what makes the replication terminate when the polymerase reaches the primer at the other end" since when you perform a PCR you go through three phases. The ...
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4 votes
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Real time PCR normalization algorithm

First step is the calculation of efficiency, denoted by lets say $E_{gene}$. See this post for calculation of primer efficiency. So the fold change for that gene will be calculated by $E_{gene}^{-\...
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4 votes
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A question related to qPCR analysis

OK, let us start from the beginning. We know what makes one cell type different from another cell type is its expression - i.e. what genes are actually being transcribed into RNA. Therefore, there ...
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4 votes

DNA barcoding and real-time PCR

You've done a pretty decent job of answering your own question, but there are a few things that can be elaborated on. The general convention is to use the mitochondrial Cytochrome C Oxidase 1 (COI) ...
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4 votes
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DNA extraction methods for hair?

I've been trying to figure out something that works for some time. I have recently tried 3 cheap protocols using commonly available lab reagents. I was using buccal samples (0.2 ml of spit). 1) ...
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4 votes
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What happens if both forward and reverse primers have same Tm?

It is actually good if both the primers have the same Tm (Melting Temperature) because you would basically want both primers to anneal at your ...
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