It is not completely coincidence that the samples that amplify well have no PD and the ones with strong PD have not amplified. The pd may look faint but its size is small so it traps very little EtBr so is faint but there is lots of it. For a given primer pair if the quality of the dna is good it amplifies well from the start and uses a lot of primer being incorporated into pcr product. If the dna is poor quality ( or contains inhibitors) then it does not amplify well and the enzyme tries to do something and as extension is hundreds of bases a second even the smallest binding of 2 primers will extend, After that the PD melts easily and has perfect homology with the primer so its amplification is very efficient and eventually it removes much of the primer so good pcr product cannot form.. The ability of primers to dimerise is minimised by using good design parameters and software like Primer3. Adding too much primer to the reaction mix can make PD more of a problem

Dear Cristian Ricaurte ,

To more fully answer your question I would ask for some aspects of your assay:

One important question:

- you mentioned you are using the same concentration of primers. Are you amplifying the same sequence or different sequences (i.e. are you investigating different genes? Or are you just checking for the amplification of a sequence in different samples?

If you are checking for different sequences:

- do all these pairs have similar biochemical and physical-chemical properties? If not, you may have to optimize a primer concentration for each primer pair;

- do any of these pairs have a very negative deltaG for the formation of homo- or heterodimers? If you have not taken into account primer-dimer formation during primer design, this can be an issue in your experiments.

If you are checking for one pair of primers in different samples:

- First verify if dimer formation is not too stable, as mentioned previously;

- Verify if dimer formation is not stable in the amplification step of the PCR (at that temperature, to be precise);

- Choose a concentration of primers, in which little dimer formation is observed. If you do not observe amplification in some samples, but do in others without primer-dimer occurrences, you may have a distinct expression of your amplicon between your samples.

Hope this helps.

It is not completely coincidence that the samples that amplify well have no PD and the ones with strong PD have not amplified. The pd may look faint but its size is small so it traps very little EtBr so is faint but there is lots of it. For a given primer pair if the quality of the dna is good it amplifies well from the start and uses a lot of primer being incorporated into pcr product. If the dna is poor quality ( or contains inhibitors) then it does not amplify well and the enzyme tries to do something and as extension is hundreds of bases a second even the smallest binding of 2 primers will extend, After that the PD melts easily and has perfect homology with the primer so its amplification is very efficient and eventually it removes much of the primer so good pcr product cannot form.. The ability of primers to dimerise is minimised by using good design parameters and software like Primer3. Adding too much primer to the reaction mix can make PD more of a problem

Hey...are u isolating plasmid by kit or by mannual method?

Sagar Baravkar Hi Sagar ! I'm nos isolating a plasmid is total bacterial DNA.

Paul Rutland Hi Paul, thanks for your answer. I look for absence of amplification in the samples that have primer dimer formation. But do you have any idea about why in some of those samples where I'm not looking for amplification, I see PD and in other samples I don't ? Would you suggest minimize the EtBr concentration ?

Primer dimer does not need a template dna to amplify. It is just the 2 promers sticking together where there is some homology between the primer sequences and extending with each other as a template. Changing ETBR will make no difference to PD formation. PD formation is almost always strongest in the no template control or when amplifying poor quality dna

The 3 ways to decrease PD are

1 Use a hot start Taq polymerase

2 Use less primer in each reaction

3 Design better primers