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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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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16 views

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? [on hold]

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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43 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 so ...
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51 views

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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37 views

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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60 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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93 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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91 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 Designing [closed]

I have a basic idea about the requirements in primer designing. Basically my knowledge is limited to theoretical knowledge and have no experience in actual primer designing. And also I have a basic ...
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42 views

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. ...
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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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139 views

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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46 views

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
168 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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2answers
752 views

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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562 views

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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7k views

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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695 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 ...
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248 views

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 ...