Questions tagged [pcr]
Polymerase Chain Reaction is a method of replicating DNA exponentially from even a single molecule.
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Qpcr Copy number formula
I recently started to get involved with quantitative PCR. I have some data and I want to calculate the copy number of them. In the output Excel file, I have the following cells (I am running ...
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qPCR from tissue to compare T-cell subpopulations
In the experiment, groups of mice have been intranasally immunised with different vaccine formulations. We have stored nasal tissue (basically, the front part of the skull without muscles, brain, etc.)...
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How to compare basal expression of a gene in two tissues using qPCR techniques?
I have two tissues and they are not treated; I would like just to compare the basal level of expression of the gene of interest (goi) in these two types of tissue (tissue1 and tissue2). After qPCR, I ...
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How to quantify basic levels of samples in qPCR?
I do have to quantify the difference in basic levels of expression for a given gene in two types of tissues. There is no treatment, just basic levels.
Before I had a "control" sample that I ...
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modify PCR steps to include a ligation
Imagine a multiplex PCR in which after the extension step, the dsDNA is nicked and requires a ligation reaction to repair the nick before the next denaturation step. So the steps would be:
Denature
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What should be considered a GC clamp in a qPCR primer?
Hello there!
After reading different sources regarding designing of qPCR primers, I'm a little confused regarding the concept of GC clamp.
Can you help me by telling which of these cases below is ...
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Explanation of QPCR's "mixing curves."
I just started using qPCR (learning the logic and the basics, actually). I am trying to create a standard curve for ecoli and Pseudomonas syringae. I started by making some DNA dilutions of 10%, 1%, 0,...
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PCR primer design for specific region
I want to amplify a specific region of my genome. I want the segment to be as precise as possible but the primers that would align to the start and end of the target fragment are not optimal in terms ...
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Testing primer for misspriming in 770 Mb lepidopteran genome
I designed primer pairs for a lepidopteran genome (Cydia pomonella, codling moth, ~770 Mb, ~60% transposable elements, Reference Genome Paper). I have around 60 primer pairs that bind to a specific ...
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Why do the components of PCR mixture need to be added in a specific order?
I have recently been told that the components of a PCR mixture need to be added in a specific order: primers, dNTP, and finally polymerase. May I know why this is the case?
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Linearising plasmids for CRISPR experiment
I am currently designing a mock CRISPR knock-out experiment, and I’m wanting to insert a plasmid for selection. Using a restriction enzyme at 2750bp for cutting, would the location of the cut site ...
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Has anyone ever used TC-512 gradient thermal cycler / TECHNE to optimise annealing temperature?
I’m not sure how to perform gradient PCR in order to optimise annealing temperature using this thermal cycler.
As I performed gradient calculator, I set temperature to 55 °C and gradient range to 10. ...
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TMPRSS2 Primer Design
I am trying to design a primer for TMPRSS2 PCR reaction. However, this gene is in reverse position like in the picture I'll show.
From the nucleotide sequence I got from NCBI, should I reverse the ...
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Would a size-specific, electrophoresis-based RNA quantification kit work for ssDNA as well?
I'm using a custom library of ssDNA oligos to test the performance of some qPCR assays with mismatched bases in various primer and probe regions, and I'm trying to come up with a good way to verify ...
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How to avoid mutations when performing Gibson Assembly (or generating amplicons during PCR)
More often than desired (about 75 % of the time), when building plasmids via Gibson Assembly (~ 5 kb plasmids; from a maximum of 2 fragments) we obtain clones with several random point mutations all ...
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Primers spaning exon-exon junctions
I am trying to choose which of two primers is most appropriate to use in qPCR, based on if the primer spans an exon-exon junction or not, where the primer that does span an exon-exon junction is ...
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What is the function of PCR in the whole genome sequencing?
Whole genome sequencing (WGS) method is using PCR in one of their steps. In the following page from the CDC, we find this diagram:
It says that the PCR is needed to "make copies of each DNA"...
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PCR: From transgene/microgram to cell concentration
I am doing research on CAR T-cell kinetics. The measurement of CAR T-cell concentrations across time is normally carried out with qPCR (see here, Fig. 1). These concentrations are generally reported ...
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PCR for gene expression [closed]
Could somebody please explain the basic principle behind how qPCR can detect if a gene is expressed or not? I tried looking at literature on ScienceDirect and other websites but I could not find any ...
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How do you do Polymerase Chain Reaction (PCR) if you don't have a thermal cycler?
PCR has three steps: Denaturation, Annealing, and Primer extension. Let's say you do not have a thermal cycler in the lab, how would you mimic similar conditions and perform PCR?
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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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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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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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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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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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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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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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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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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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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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