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

There are many methods of DNA isolation, so it is difficult to make broad statements about all situations. Typically, though, during the DNA isolation protocol, essentially all proteins are denatured ...
MattDMo's user avatar
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5 votes

Can primers for PCR be duplicated?

Short answer: You need to buy some more, but you need the sequence also for ordering. Long answer: The Taq polymerase needs a piece of DNA (or RNA) to prime the reaction and be able to enlarge the ...
Chris's user avatar
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5 votes

Why PCR primers don't amplify beyond the target region?

How does DNA synthesis from the forward primer stop when it reaches the point where the reverse primer is? Briefly, it doesn't. You get a lot of single-stranded long pieces of DNA (step 1 here, ...
aaaaa says reinstate Monica's user avatar
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Do the primers used for Covid-19 test work with all new strains?

I TRY to answer. First of all, no we cannot be 100% sure that the primers are effective against all strains. However, the strains that are under media attention right now are due to variants in the ...
Fabio Marroni's user avatar
5 votes

Primers spaning exon-exon junctions

See similar questions elsewhere. Looking into this paper on primer design, they write this: It is often necessary to choose the primers from the exon—exon junctions (E-E-jns), the regions of mRNA ...
Maximilian Press's user avatar
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What happens if both forward and reverse primers have same Tm?

It is actually good if both the primers have the same Tm (Melting Temperature) because you would basically want both primers to anneal at your ...
WYSIWYG's user avatar
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4 votes
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Why PCR primers don't amplify beyond the target region?

They don't stop replication. It's just that you replicate your dna in a number of cycles, let's say 30. after which you'll have a approximatly 10^6 molecules of the amplified region and only a few ...
Jonas's user avatar
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4 votes

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

No, some strains contain mutations causing partial or complete failure of amplification of at least one of the test targets. Here's an FDA bulletin on the issue from January: https://www.fda.gov/...
Armand's user avatar
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3 votes

After the primer is removed from the leading strand, how does DNA polymerase I add dNTPs without a 3'-OH?

The leading strand post-initiation First consider the DNA replication fork after initiation has occurred. The 3′-OH primer on the leading strand is the 3′-end of the strand of DNA being synthesized in ...
David's user avatar
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3 votes
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Different kind of M13 primer

This dates back to the 1980's when these M13 primers were first cloned. Heidecker G, Messing J, Gronenborn B. 1980. A versatile primer for DNA sequencing in the M13mp2 cloning system. Gene 10(1):69-...
canadianer's user avatar
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3 votes
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Gene cluster of interest not being amplified in PCR

A few things to consider: Template: Are you sure the template you want is on a plasmid, and not in the Rhizobium genomic DNA? (In which case it would not be present in DNA purified with a plasmid ...
MelaGo's user avatar
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3 votes

How different can the Tm be between 2 primers?

This really depends on your application, but from my experience 2-3 degrees with a max of 5°C difference usually works. The more the melting temperature differ, the more you need to optimize all the ...
Chris's user avatar
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3 votes

How different can the Tm be between 2 primers?

The default parameter provided by "primer-blast" is 3 degree (max difference between the melting temperatures of the pairs) . that said I think it really depends on the application. in case of cloning ...
edoardo pedrini's user avatar
3 votes
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Manual Primer Design for a gene on the reverse strand

Not a stupid question at all. I was just as confused by this as a beginner in molecular biology. You get the hang of it after a while. :) I find it much more difficult to design primers from just one ...
CDB's user avatar
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3 votes

DNA extraction for LAMP assay

Although this is theoretical a problem, in fact it isn't one. Lets do the calculation around it. EDTA complexes equimolar amounts of bivalent cations (e.g. Magnesium), so 1mM EDTA will complex 1mM ...
Chris's user avatar
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3 votes
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Detection of primer dimer on RT-PCR?

This is a bit complicated from far away, but I will share a few thoughts on it. The occurence of primer dimers depends on the design of the primers (sometimes they are inevitable due to sequence ...
Chris's user avatar
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3 votes
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Ordering Primers: When to choose wet vs dry delivery format?

Pros of wet: No need to rehydrate to use. For e.g. 96-well plates, this can be a big deal as it's a pain to do this for large numbers of samples. More accurate concentrations (oligo manufactureres ...
Maximilian Press's user avatar
3 votes

Hierarchial and shotgun sequencing vs Massively Parallel Sequencing

The construction of sequencing libraries for Illumina, PacBio, ONT, or similar instruments is a large field of methodology that encompasses a variety of different techniques. For background on ...
Maximilian Press's user avatar
3 votes

TMPRSS2 Primer Design

If your goal is simply PCR amplification than it makes no difference whether you flip the sequence or not: By convention the 'forward' primer refers to the primer that is designed against the anti-...
Risk Complex's user avatar
2 votes

DNA extraction for LAMP assay

The conventional Eiken LAMP formulation has an excess of Mg2+. 5 µl of TE with EDTA at 1 mM going into a 25 µl reaction will have minimal impact. In any case the LAMP formulation can be up adjusted ...
whatbox's user avatar
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2 votes
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Which is the reference 16S rRNA?

As you correctly point out, designing an optimal primer pair for 16S-rRNA sequencing is a tricky affair because even the less-variable regions are not same between different strains and species. Sambo ...
WYSIWYG's user avatar
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2 votes
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How to design internal primers?

If you are not getting amplification, there are multiple things to visit before you want to design internal primers. You should try troubleshooting your PCR reactions before designing and ordering ...
Anurag Mishra's user avatar
2 votes
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CRISPR guide RNA design and primer synthesis

A quick check with BLAST returned the following result: ...
alec_djinn's user avatar
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2 votes
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Meaning of "primers IL-2" in a scientific article

From the description, yes, those primers are supposed to be specific for the IL-2 gene. The way they would be used would be on mRNA, not DNA, so you could use them to quantify the level of IL-2 ...
iayork's user avatar
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2 votes
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Sequencing inaccurate at the primer site

The very extremities of sequencing reads obtained by most if not all sequencing technologies are usually of lower quality, though more often so in the 5' region. You should disregard this data, or ...
Joe Healey's user avatar
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2 votes
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qPCR: do both primers have to contain a GC-clamp in order to be effective?

You can have GC-clamps (3' terminal G/C) in both primers; this may overall improve the PCR efficiency. However you should take care not to put too many ...
WYSIWYG's user avatar
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2 votes

probability of hairpin vs self-dimer formation?

I have tested a similar design to this. With a duplex protocol the chance of forming a hairpin is much higher than that of forming self-dimer, even at concentration of 25 μM. You can run a 3-5% ...
Jianli Li's user avatar
2 votes
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How do primers anneal to ssDNA?

No enzyme is involved in base-pairing (annealing). This does happens by chance, but the chances are governed by stoichiometric considerations. The main such consideration is that the (low molecular ...
David's user avatar
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2 votes

PCR markers for C57B6

Jackson Labs offers a service for this for what looks like a reasonable price. I have no experience with the service other than general good feelings about Jackson. We have a unique selection of ...
iayork's user avatar
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2 votes

Why don't we use hybridization instead of PCR?

From comment. Could you be more specific about what you mean by "hybridization", e.g. Southern blot? This is the old-fashioned technique that I believe is closest to what you mean. Slot ...
Maximilian Press's user avatar

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