Questions tagged [primer]
A primer is short oligo made from nucleic acids. It is the starting point for DNA polymerases to start DNA synthesis. DNA polymerases can only synthesize enlarge existing nucleic but not generate new ones 'de novo'. Primers can be made of DNA (for PCR) or RNA (naturally occuring).
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Are the Tm and Ta values of PCR primers based on entire sequences or just annealing portions?
I am trying to use the Q5 mutagenesis kit to produce an indel mutation in my protein.
I used the NEB BaseChanger interface to design my primers (https://nebasechanger.neb.com/). However, I am confused ...
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Primer dilution verification
I just received a primer (details are displayed in the picture). Since this is my first time doing this, I want to do it correctly. My goal is to make a 10µM primer.
So, If I get it right and based on ...
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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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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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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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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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Primer design for site-directed mutagenesis
In our practical course about modern cloning methods, we performed point mutations on a promotor via site-directed mutagenesis.
As far as I understand that method you need forward and reverse primers ...
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Hierarchial and shotgun sequencing vs Massively Parallel Sequencing
Why is the cloning step unnecessary prior to sequencing with massively parallel sequencing (MPS; Illumina or similar technologies), when it is necessary for hierarchical and shotgun sequencing?
I know ...
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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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Ordering Primers: When to choose wet vs dry delivery format?
Whenever ordering primers, I'm always asked to specify which delivery matrix I prefer to have the oligos delivered in: wet (in water or some kind of TE buffer) vs. dry.
My understanding is that when ...
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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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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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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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How to calculate the occurrence of a stretch of nucleotides in a genome?
I have seen that the formula to calculate the number of times a given sequence of nucleotides occur in a target genome is derived from that to calculate the expected frequency of restriction sites:
<...
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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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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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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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Design primers for PCR from given DNA sequence
Given the sequence: 5’-ACTGACTATGTAGAA………GGCCCTAAGGGCCAA-3’ (1)
You wish to do PCR of this dsDNA to add “overhangs” on the ends of it. On the 5’ end of you will put
the restriction site for ...
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design and assessment of qPCR primers for COVID19
I read the False Negative of qPCR test for COVID19 is high compare to CT scans.
https://pubs.rsna.org/doi/10.1148/radiol.2020200642
I was curious if experts can comment on why and what makes it ...
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PCR markers for C57B6
Do you know any primers that can be used to genotype mice and check if they are still C57B6? I'm concerned about genetic drift in my colony. I bought a breeding pair 3 years ago and expanded. I wanna ...
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Can primers for PCR be duplicated?
Complete beginner question here, don't laugh:
If I have some primers that have been synthesized, and I am close to running out of them, is there any way to duplicate them / amplify them / synthesize ...
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How do primers anneal to ssDNA?
In a PCR type protocol, the high temperature stage of the cycle will cause dsDNA to denature, as well as any annealed primers. At the lower temperature the denatured dsDNA will remain denatured. As ...
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probability of hairpin vs self-dimer formation?
I have a linear ssDNA oligo that is designed such that the ends are complementary, and conducive to hairpin formation (see attached figure). My question is, which has a higher likelihood of formation, ...
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Gene cluster of interest not being amplified in PCR
In a lab, I currently have a sample of Rhizobium sp. NT-26. This bacteria is a chemolithoautotrophic arsenite-oxidizer, and I want to clone the arsenite oxidase genes into another bacteria strain in ...
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Primer design for PRP24-TAPtag
I have a problem with my proteomics homework. I was asked to design primers used to create a homologous recombination box to create PRP24-TAPtag yeast. I know that homologous regions should be 40-50nt ...
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How different can the Tm be between 2 primers?
I have a forward primer with Tm 85 degree Celcius. I cannot change this primer. But I have two options for the reverse primer. One with Tm 65 degree Celcius (Annealing at 65 degree Celcius) and ...
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Sequencing inaccurate at the primer site
The times I have sent a sample to sequence, both the forward and the reverse primer sites, show high inaccuracy while the rest of the gene is correctly sequenced. Because of this, the sequences of my ...
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double PCR with tailed primer
I would like to make a double PCR with universal tailed primer. The first PCR would make a tailed amplicon. The second PCR would generate fragment labeled with fluorescent dye. Here is my protcole. ...
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After the primer is removed from the leading strand, how does DNA polymerase I add dNTPs without a 3'-OH?
I have a question about replication in prokaryotes. I learned in school that:
DNA polymerase needs 3'-OH to add a dNTP.
The chromosomes of prokaryotes are usually circular.
The primer in the leading ...
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Different kind of M13 primer
I m looking for universal primers M13. I found different version. For instance :
M13 (-21) Forward TGTAAAACGACGGCCAGT
M13 (-40) GTTTTCCCAGTCACGAC
What's the meaning of (-21) and (-40 )
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In PCR what is the chemical makeup of the primer? DNA or RNA? [closed]
I'm thinking the answer is RNA. Is that right?
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Specific primer design
I'm designing primers for species-specific amplification (inside ITS1-ITS2 region). The problem is that when I blast (GenBank) my primer pair, the Forward primer anneals with some isolates (of the ...
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How does a polymerized TruSeq adapter affect sequencing reads?
I am trying to build a NGS library on enriched DNA (DNA length = 93 bp). I am using PCR amplification with two overhanging primers, both containing Illumina adapter sequences (Universal Adapter & ...
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CRISPR guide RNA design and primer synthesis
I am trying to knockout huntingtin gene from HeLa cells (human epithelial cells). I used CRISPR explorer and benchling.com to determine the best guide RNA sequence that would bind to the target DNA ...
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Meaning of "primers IL-2" in a scientific article
From an article ("Amelioration of Experimental Autoimmune Encephalomyelitis in Lewis Rats by FTY720 Treatment", 2003):
We performed PCR amplification in a 100-μl reaction mixture containing 200 μM ...
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Manual Primer Design for a gene on the reverse strand
My question might sound very naive and stupid but I am hopeless now. I read so many websites and pages but could not figure out this PCR primer design thing completely.
Some genes are on the reverse ...
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Designing degenerate primers using alignment of protein sequences from other species
I am trying to design a PCR primer for a gene whose sequence is not known. Even the whole transcriptome sequencing done in our lab did not identify that particular gene. Hence, I guess I am left with ...
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Designing primers with restriction sites
I want to design a forward and reverse primer that include overhangs with restriction sites, with the dna used for restriction enzyme cloning.
If I want to send my dna to a synthesis company should ...
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How to design internal primers?
I have sequences of the Cytochrome c oxidase I (COI) from several populations of Peringia ulvae (Mollusca; Gastropoda; Littorinimorpha; Hydrobiidae). I need additional COI sequences from a specific ...
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Why are two forward and reverse priming sites depicted here? what do they do?
This question is in regard to the baculovirus expression system.
(source: amsbio.com)
Why do we need to generate primers for the polyhedrin promoter and the baculovirus?
Primers are needed to ...
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Which is the reference 16S rRNA?
Recently, I've stumbled upon a fact, which hasn't bothered me for many years. The fact is that all universal 16S primers are written as "[FR][0-9]+" (in regex notation), that is they have a position ...
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Is a Linker sequence mandatory in primers for PCR Cloning?
I have designed the primers for PCR cloning, but i was not getting any colonies after the ligation. One of our colleagues said, that the restriction digestion might not be working as there is no ...
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If we add only 1 primer in PCR [closed]
What would happen if we add only one primer, say forward primer, to PCR?
(Image Credits: Wikipedia) As it is clear from the image that we need both forward and reverse primers to get it working (...
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Optimum gibson assembly overlap length
Does anyone know the optimum length of overlap your primers should have to your vector for gibson assembly. Most manufacturers say to do at least 20 bp on both side but don't give a maximum. Sometimes ...
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qPCR: do both primers have to contain a GC-clamp in order to be effective?
I'm developing software which, among other things, designs primers pairs (forward and reverse) for qPCR. In my research on various properties, I read about GC-clamps and I understand the basics of it ...
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What happens if both forward and reverse primers have same Tm?
One of the key rules in primer designing is that Tm (melting temperature) of forward and reverse primers should be in the range of ±5°C. For example, if ...
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DNA extraction for LAMP assay
I am trying to set up a LAMP assay (Loop-mediated isothermal amplification) for the detection of E.coli.
I was told that EDTA can chelate the Mg in my reaction and thus prevent the assay from working ...
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What are flagged primers? [closed]
I'm interested in amplifying a sequence for further use with Gibson Assembly. I want to create overhang regions in my DNA fragment so there would be complementarity to the plasmid I'm trying to insert ...
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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. Why ...
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