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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 ...
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

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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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
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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 ...
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, ...
5 votes
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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 ...
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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5 votes
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What is the function of PCR in the whole genome sequencing?

PCR is used to replicate each isolated genome fragment, yielding several copies of each fragment in your DNA solution (that‘s called a library = a collection of fragments + adapters). This amplifies ...
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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 ...
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) ...
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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4 votes

PCR Mastermix Preparation

A PCR Master Mix is just a way to speed up your pipetting. Instead of preparing 10 different reactions of 20ul each (for example) you prepare one reaction of 200ul and then you split it into 10 tubes. ...
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4 votes
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Why PCR primers don't amplify beyond the target region?

They don't stop replication. It's just that you replicate your dna in a number of cycles, let's say 30. after which you'll have a approximatly 10^6 molecules of the amplified region and only a few ...
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4 votes

Do the primers used for Covid-19 test work with all new strains?

No, some strains contain mutations causing partial or complete failure of amplification of at least one of the test targets. Here's an FDA bulletin on the issue from January: https://www.fda.gov/...
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4 votes

How to assemble three 60mer nts by pcr?

In order to assemble your TAG oligos by PCR you will need to redesign your primers so they are complementary to the TAG oligos as DNA polymerases work by adding nucleotides to the 3' end of a DNA ...
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4 votes

How are PCR tests for detecting COVID-19 performed on hamsters?

This is probably quite different from the example you're thinking of, but here's an example where they were using a hamster as a model organism for testing possible COVID-19 treatments for humans: ...
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