Hi Francesco,

It looks like you have a very intense and small PCR product.

I don't know what DNA stain you are using, but they are positivly charged, while the DNA is negetivly charged.

They will migrate in opposite directions under electrical potential: as the DNA moves from the top to the bottom, the DNA stain moves from bottom to top (the bottom of a gel will have little or no stain and therefore appears dark).

That small intense PCR product is binding the DNA stain as the stain moves from the bottom to the top of the gel (and is also binding the stain out of the gel as the PCR product moves from top to bottom).

Loading high DNA concentrations into a well and running it will produce the smearing commet like tail from the PCR band. Try loading much less (1/10) and see what happens.

Best Wishes,

Robert.

Greetings,

Well, I think i can see the same small band in other Wells, but fainter. Anyway, something Like this happened to me when I was trying to amplify a 500 pb or so gapdh amplicon. Check your primers for secondary structures or.primer dimer formation kinetics, Delta G should be higher than -10 to avoid primer dimer formation. I almost can assure you that if you double the DNA concentration in that RX, the smaller band will decrease, and the bigger one increase, as this effect magnifies when DNA concentration is low. You could be wondering about, "why do this happens only in one of my vessels", and there could be many answers to that, however, it's most likely to be a low number of copies or even DNA degradation (to some extent), that is making that inter primer interactions are favored against gDNA amplification. Also, check for possible inter-amplicon regions that might be causing primers to Miss align.

Good luck.

Dear colleagues, I agree with your ideas, but we should consider and "mechanical" troubles. Sometimes you can receive similar band as a result of some troubles with a wells in gel. Or it could be extra volume of your sample at start. I saw simillar results. Of course, it is only my advice, but try again with the same sample in new gel, please.

Adán Valenzuela Castillo thanks for the contribution, I guess the faint bands are higher than the very bright one. I am not interested specifically in the other bands, but my question is more related to the extra-dye looking-like shape of the sample number 11 from left (excluding the marker).

Robert Mulhall I am currently using 6x loading dye for DNA, from ThermoFisher, while the gel stain is the ECO safe DNA gel stain from Pacific Image. I am using it since a month and I am not having any trouble since.

I have seen similar smearing when I've ran high DNA concentrations. This is easily solved by using less temple with the loading dye.

If there is still smearing after you dilute down your amplified DNA, then the gene target may be atypical - try using a different primer pair.

Andrei S. Babenka I indeed think about a personal mechanical/human mistake here. I do not see why only one sample would have such a bright and intense color.

I was checking the compatibility between DNA loading dye and gel dye but it seems ok to me.

I will repeat the experiment with lower concentrated DNA as suggested by Robert Mulhall .

Thanks for the suggestion, I hope to learn from my mistakes!

Francesco Desiderio good luck. it is a very interesting to know the result, please upload it if you can. Thank you.

Hi all,

I increased the number of cycles (35 to 40), decreased the Gel Dye concentration (1/3) and increased by 1 Celsius the Ta. At the moment what I have is the disappearance of the strange lower band.

I have moved the position of my samples but after the marker, you might look at sample in position 10 (where marker is 0) and observe still a brighter band but at the same position as the previous.

Best

Hi,

It's most likely to be because of the temperature change and the longr run, as it should be increasing the target amplicon - unspecific by-products ratio. As I told you before, the latter must had been dulling your target band. Great to hear that you got ir right this time. Cheers!

Hi,

It's most likely to be because of the temperature change and the longr run, as it should be increasing the target amplicon - unspecific by-products ratio. As I told you before, the latter must had been dulling your target band. Great to hear that you got ir right this time. Cheers!