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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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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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597 views

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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Adjust amplicon size with universal primers

I would like to create primers with universal primers M13. 5'-(Universal seq) - (Adjust) - (Specific Primer)-3' What sequence do I need to (Adjust) primer ...
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231 views

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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Real time PCR for amplification of long amplicon

As far as I know, the ideal PCR product (amplicon) length should be ranged 100-300bp for doing real-time PCR. However, I got some primers that will be generated products in 900bp and 1500 bp, I want ...
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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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Can I modify the RPIX primers for MiSeq Sequencing?

I am in the process of making a 48 sample pooling library prep, and I am about to order the RPIX primers for Illumina MiSeq sequencing. The primer is like this P7 - i7 Tag - UNKNOWN - RA3 example ...
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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. http://www.amsbio.com/images/pagelayout/BPE.jpg Why do we need to generate primers for the polyhedrin promoter and the baculovirus? ...
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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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435 views

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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2answers
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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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135 views

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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670 views

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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1answer
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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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1answer
3k views

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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3answers
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Does common PCR amplify genes regardless of what cells / barriers they are in?

I have some understanding of how PCR testing works. What I have always been wondering: how can we be sure that a primer reacts with the targeted gene(s) regardless of where¹ the genes are inside a ...
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Is there a good site or software to see if a primer pair spans an exon junction?

I am going to perform some RT-qPCR tests to validate an experiment.  I'm currently in the process of ordering primers, and I would like to get them from the Harvard Primer Bank since these have been ...
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1answer
132 views

Primer Design with Primer-BLAST over specific site

I am trying to design primers using Primer-BLAST such that the forward primer spans a specific base pair site. I am looking at KRAS for which I believe the RefSeq ID is NG_007524.1 and the forward ...
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1answer
367 views

Design arbitrary degenerate primers (with non-binding criteria)

I would like to design a number of arbitrary degenerate primers (primers with variable bases, e.g. NGATWGCTSATNGC) for a TAIL-PCR. I would like to be able to ...
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1answer
1k views

Which pair of primers should be used to amplify the ORF in PCR? [closed]

So I want to choose the correct set of pair of primers to amplify the ORF of the gene that corresponds to amino acids in a protein. The start and stop codons are underlined. (I know that these need to ...
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Primer heterodimer problem

I design my primers in Primer3PLUS and analyze them in Oligoanalyzer3.1, A big problem that I have this is that I got very nice output in NCBI-Primer BLAST for their specificity but a very negative ΔG ...
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Primers for human tissue? [closed]

If I wanted to examine human tissue samples, such as hair, blood, fingernails, etc, for their DNA, what primers would I use for the amplification of the DNA when extracted(PCR)? edit: To look at the ...
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Multiplex PCR, shorter amplicon inhibiting longer amplicon?

I want to run multiplex PCRs for my genotyping, with a primer pair targeting my construct and a primer pair targeting some housekeeping gene (sort of a built-in control). I designed the control ...
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Primer design for HLA locus

i have designed primers for HLA locus DPA1(exon 2 region) based on Real-Time PCR (qPCR) Primer Design guidelines. primer will start from intron regions to cover full exonic region. F-...
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1answer
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Does DNA replication in 5' to 3' (leading strand) need RNA primase?

https://www.youtube.com/watch?v=27TxKoFU2Nw In the above video it shows that during DNA replication, the lagging strand require RNA primase to add 3' -OH group for further addition of nucleotides. ...
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What are possible reasons for a DNA template not giving amplification?

I am trying to amplify ~10kb length fragments using NEB LongAmp Master Mix. I had been using one particular aliquot of DNA (template amount in each PCR reaction of 10ul being around 60ng) and getting ...
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Degenerate primer design for DIG in situ hybridization

New to molecular and have learned to design primers from google/youtube so any info would help Would someone be willing to share their protocol for degenerate primer design? Breakdown: Trying to ...
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1answer
238 views

Real-time PCR delay in Cq due to insertion SNP in primer

I am collecting evidence, even anecdotal, how does single nucleotide deletion or insertion in primer region affect the outcome of real-time PCR. I am most interested in how much there is a delay in ...
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How does sequence independent single primer amplification work?

I'm having difficulty finding an explanation of how single primer amplification of DNA works in the literature available to me, can anyone explain the methodology and what it accomplishes?
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Effect of single nucleotide deletion or insertion on primer annealing

How is primer annealing, and, consequently, PCR amplification affected by single nucleotide deletion or insertion inside the primer ? Imagine a primer like this: GCGTCATAAAGGGGACGTG (primer) and ...
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3answers
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When designing primers how important is the GC clamp?

I'm designing a set of primers and reading about the principles of primer design one of which is: GC Clamp: The presence of G or C bases within the last five bases from the 3' end of primers (GC ...
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1k views

Primer Dimer / Hairpin Algorithms

What are the algorithms / methods in use for the calculation of primer dimers and hairpins? As an example, IDT's OligoAnalyzer tool will generate these analysis given particular sequences. The homo-...
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How can you identify if a person is homozygous for a certain allele?

I've been thinking about starting a small private research project. In this project I need to find out whether a person is homozygous for a certain allele. The reason for this is that I'm really ...