Questions tagged [pcr]

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

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Why does plasmid cloning efficiency go down with increased size of insert?

I am setting up a cloning experiment. Briefly, I ran PCR on a gene of interest, cloned it into a vector (pGemTeasy) and bulked it up in bacteria. I have been advised to limit the size of my fragment ...
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Biology (DNA electrophoresis with agarose)X

I'm researching a polymorphism using restriction fragment length polymorphism (RFLP) and gel electrophoresis. After RFLP, I should see fragments at 141bp and 111bp, but I can not see in 2% agarose, ...
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Datasets for Polymerase Cycling Assembly

I am currently studying Polymerase Cycling Assembly. I would like to find examples (or datasets) of sets of oligonucleotides that are effective for PCA. It would also be good to know of sets that ...
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How precise does a 1:1 ratio PCR sample mixture (microliters) have to be for the liquid amount ratio to not have any effect on results

Amount of liquid: 5 micro liters master mix 5 micro liters of patient sample
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Do 0.2 vs 0.5 ml epitubes require different PCR machines, incubators, etc?

Eppendorf themselves and many other companies list on their website two above mentioned sizes of "PCR tubes". As I understand 0.2 ml is by far more widespread and "standard" for ...
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How are PCR tests for detecting COVID-19 performed on hamsters?

This is something I've been wondering about ever since the stories of people finding hamsters with a COVID infection in Hong Kong. Apparently those animals were tested using a PCR test, but I'm ...
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qPCR - different results total RNA vs mRNA

I have performed qPCR on a tissue, where I have extracted total RNA and also purified mRNA. I ran the qPCR samples together, and have therefore been exposed to the same conditions except the ...
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How many non-pairing bases can a PCR primer have in directed mutagenesis?

I'm not including a lot of details here, because the problem I'm working with is actually more complex. Say you have the DNA sequence 5'-...tct gcg gtg gtt ggc att ctg ctg...-3' Could this sequence ...
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Is there any methods to avoid the pcr control contamination?

Why do I use the primers in the D1-D2 region to amplify the fungus, and using water as a negative control has a weak band that is the same size as the target fragment? When using the ITS primer, using ...
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Transformation and PCR in molecular cloning

After obtaining the recombinant DNA, it is common to transform into E. coli to screen for recombinant DNA and amplify it. But I would like to ask can we amplify it using PCR? I think there will be 3 ...
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In what sense is PCR a "nuclear-derived" technique? [closed]

The International Atomic Energy Agency (IAEA) has launched an initiative, ZODIAC, to combat pandemics that originate in animals. In part, this involves the deployment of kits utilising real-time ...
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Why don't we use hybridization instead of PCR? [closed]

So, I would like to ask, why don't we use just DNA hybridization instead of PCR primer amplification to diagnise some illness? I know, when you have a really small amount of DNA from virus or cell you ...
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Lowering annealing temperature in PCR

In the book "Lewin's Genes" there is the following quote: Lowering the Tm of a PCR reaction—in effect, relaxing the reaction stringency and allowing primers to anneal to not quite perfect ...
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How did we do the COVID PCR after its first appear?

I recently learned about the PCR testing and its idea of using primers to replicate the DNA or RNA. By this I assume that we need to know the sequencing of the DNA to make our primers. If that is true;...
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Using PCR to add the overhangs to gBlocks for NEB HiFi

Does adding the overhangs to gBlocks for NEB Hifi using PCR pose a big problem? The IDT website suggests that one should design the gBlocks with the overhangs. However, if I did not do this, will it ...
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The average of Ct values in real-time PCR

The Ct value is the number of cycles when the "PCR amplification curve with the horizontal axis as the number of cycles" and the "predetermined threshold" intersect, and is not ...
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3 votes
1 answer
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Polymerase Cycle Assembly (PCA) is not working (Only primrs in EF Gel)

Helllo All 👋! I am working trying to assemble dsDNA fragments (for further golden gate domestication) via polymerase cycle assembly (PCA). After designing the required oligonucleotides with overlap ...
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Unknown Issue with qPCR Standard

I am currently doing the ground work for a SELEX experiment and I am attempting to verify my standards for the starting library. I ran a reaction with samples containing 50, 5, 0.5, and 0.05ng of my ...
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What are the causes of gene amplification in archaea?

I was studying this article about Genome Sequencing of a Genetically Tractable Pyrococcus furiosus Strain Reveals a Highly Dynamic Genome in order to try to extrapolate some features that could ...
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Detection of primer dimer on RT-PCR?

I usually do RT-PCR test for covid-19. Curves for Patients and Positive CTRL mostly appeared at 20 to 25 Ct. Sometimes, there are some curves that emerge at the last 5 cycles. Some argue that they are ...
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2 answers
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Why there is Cp/Ct (crossing point) value for negative sample in COVID-19 RT-PCR?

I am from IT/ Engineering background and have some confusion in RT-PCR. So far my understanding we extract RNA from test specimens and transcribed it into complementary DNA (cDNA), and inside PCR DNA ...
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Which method of gene amplification for toehold switches?

My team and I are from a high school and are planning to carry out some research investigating some toehold switch riboregulators which we have designed in silico. However, we have little experience ...
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2 votes
1 answer
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Is it possible to use two forward primer and one reverse primer?

I have eGFP and the gene fragment i assembled by pcr.The next step is that i have to join my fragment fragment into 3'p end of the eGFP.I have three primers FP1 is forward primer for eGFP, FP2 has ...
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3 votes
1 answer
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Is it possible to use PCR to test for Machado-Joseph disease?

Machado–Joseph disease (MJD) is a rare inherited neuromuscular disease that is caused by a mutation in the gene ATXN3. The protein encoded by this gene contains "CAG" repeats in the coding ...
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Does the false positive covid-19 PCR % referred to by Surkova & Nikolayevskyy in The Lancet mean % of all tests, or % of positive tests?

In False-positive COVID-19 results: hidden problems and costs it is said: The current rate of operational false-positive swab tests in the UK is unknown; preliminary estimates show it could be ...
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SARS-COV-2 replication speed

What is the speed of replication of SC2? Any information, including in vitro data would be appreciated. I would be interested to know the length of SC2 eclipse period, latent period and something like ...
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Nomenclature of substrates for DNA synthesis

I have read in my school textbooks that both deoxyribonucleoside triphosphate and deoxynucleotide triphosphate are used in DNA Replication as substrates. However, it is unclear to me whether the terms ...
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4 votes
2 answers
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How does PCR mutagenesis add restriction site near the gene of interest?

I have been learning about PCR mutagenesis to add restriction site right next to the gene of interest using a primer that's attached with single stranded restriction site (first image). I have drawn ...
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2 votes
2 answers
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How to assemble three 60mer nts by pcr?

Good morning, I am new to molecular biology. The question might be silly but i would like to know the answer. I have three 60mer single strand synthetic oligonucleotide. Namely Tag 1 - 3. My goal is ...
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9 votes
2 answers
247 views

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

We keep reading news of new Sars-CoV-2 strains, some of them allegedly able to evade vaccine-induced Igs. That's not unexpected, specially for a single-stranded RNA virus. However, it seems to me that ...
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1 answer
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Gibson assembley for small fragments?

I am new to the Gibson assembly, I know how I need my plasmid but main problem is I don't have short tags and linker so I wanted to do Gibson assembly? 5'-...
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VDJ sequencing in mice, DNA or RNA?

I am wondering if anyone who is well versed with VDJ sequencing for TCR repertoire analysis (specifically CDR3) would know if DNA or RNA is a better starting material? We are looking at the effects of ...
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3 primers to know if Homozygous, Heterozygous or WT

In this imaginary experiment, I am using T-DNA to make an insertional mutagenesis that will get randomly inserted into the DNA where it might get inserted within a gene, disrupting it. In order to get ...
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PCR and large-scale PCR

I have performed PCR with 50 μL, which showed a clear band in the gel electrophoresis. I then performed large-scale PCR with 3650 μL, which showed an unclear band in the gel electrophoresis. What can ...
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Can Taq polymerase be stored with PCR primers?

I don't think it's possible for TAQ to be stored with the primers, but I'm not sure. This storage wouldn't be long-term (a few days, or a week at most). Thoughts?
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What is an "open system" when it comes to PCR machines?

I have heard the term "open systems", such as here when it comes to RT-PCR machines, but I'm not 100% sure what this means, does this mean machines you can purchase yourself? Such as this ...
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What is the significance of running an uncut plasmid on electrophoresis gel?

What is the significance of running an uncut plasmid on electrophoresis gel? In this case we are talking about inserting a gene into plasmid, which then goes under PCR and then electrophoresis.
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Are the controls for RT-PCR the same as those for RT-qPCR?

I am searching for negative and positive controls for RT-PCR but all the results seem to point towards RT-qPCR. Are the controls the same for both? I have found -RT control No template control ...
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1 answer
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Does "RT-PCR" denote "real-time PCR" or "reverse transcription PCR"?

I often find the term "RT-PCR" used in articles without any further qualification. Searching for the meaning of the acronym sometimes leads to "real-time polymerase chain reaction" ...
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3 votes
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Why is my DNA band bulging?

This is the only image my the TA was able to get for us. And, we're using it for our lab report. The image isn't even ours. It's another group's image that we're sharing. But I don't understand why in ...
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5 votes
1 answer
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Is there a biological or technical reason for a 40 Ct cutoff in nucleic acid detection assay?

I have developed and validated a modified a nucleic acid test (NAT) for SARS-COV-2 detection using real-time RT-PCR (aka rRT-PCR, aka RT-qPCR). My assay is not for diagnostic use, but for donor ...
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Polymerase Chain Reaction Specifics

While going over PCR in my biology lecture this week I have come across a few questions I have about this process. First, since PCR focuses on trying to replicate a specific targeted DNA sequence many ...
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LAMP (isothermal amplification), hydroxy naphtol blue , disodium salt" or just hydroxy naphtol blue?

I am reproducing this work on LAMP (isothermal amplification) Article A new visually improved and sensitive loop mediated isotherm... , but I am a bit confused about the difference between "...
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-1 votes
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Why PCR primers don't amplify beyond the target region? [closed]

I have been doing PCR for a while. I spared a bit of time to look it up but could not find an answer. So, for sanger sequencing, if we have a primer, it can amplify about 800-1000 bp. But, if you ...
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3 votes
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RT-PCR: Not seeing how it can measure mRNA expression levels

From my understanding, in RT-PCR we start off with an mRNA molecule, use the enzyme reverse transcriptase to create a cDNA copy (hence creating a double-stranded mRNA / cDNA hybrid), degrade the mRNA, ...
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Meaning of "standard reactions" in a DNA extraction procedure description

From a DNA extraction procedure description (an in-house pharma document I'm translating into Russian): Preparation of Standards All the standard reactions should be prepared at least in duplicates. ...
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Meaning of "optical" in "Optical cap, 8X Strip" (used in qPCR)

I'm translating an English document that lists equipment used in a qPCR procedure: Reagents/Materials Optical cap, 8X Strip I googled and found that the meaning of this line is "a strip of 8 ...
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High concentration of primer in PCR

Why is it that higher concentration of primers than the DNA template in PCR will favor the annealing of a template to a primer?
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1 answer
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Humic Acid and PCR

Quite a few papers claim humic acid is an inhibitor of PCR reactions. I understand this is true when working with soil microbes, but how does it qualify to be a PCR inhibitor in general (i.e when not ...
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0 votes
1 answer
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Are specific primers or detectors, or both, used in COVID-19 tests?

I am trying to learn about the rRT-PCR testing procedure used to test for COVID-19, but I am slightly confused on one point. Are highly specific primers used with a non-specific detector, or are ...
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