Hello,

There might be several reasons for that:

- Non-specific priming and extension. This can be managed by reducing the concentration of primers in the reaction, using new primers, changing Ta conditions and reducing the number of cycles.

- Too much of the enzyme in the reaction (as well as addition of some protein factors for increased PCR processivity) may also give you some smear and even discreet faint bands that migrate slower than the specific product. This can be easily managed with purification Kits or phenol/chloroform extraction.

- Inaccurate loading of the sample onto the gel when some material is spilled out and then, it slowly reenters the gel. The use of proper tips usually helps to avoid this problem.

Good luck

Dear Mohamed,

There are three possible causes for this:

1) Contamination

2) Wrong DNA template or primers

3)Different gene isoforms

My best regards,

Muhammed

Hello,

There might be several reasons for that:

- Non-specific priming and extension. This can be managed by reducing the concentration of primers in the reaction, using new primers, changing Ta conditions and reducing the number of cycles.

- Too much of the enzyme in the reaction (as well as addition of some protein factors for increased PCR processivity) may also give you some smear and even discreet faint bands that migrate slower than the specific product. This can be easily managed with purification Kits or phenol/chloroform extraction.

- Inaccurate loading of the sample onto the gel when some material is spilled out and then, it slowly reenters the gel. The use of proper tips usually helps to avoid this problem.

Good luck

Again as other researchers have suggested there are many reasons. One of the reason I have observed previously is the method of DNA isolation. Just purify your DNA before PCR and purify your PCR products and load the gel carefully you will not get any faint bands.

Well, the first thing that comes to mind is primer dimers which are unspecific binding of the primers to other products. Are you using degenerate or promiscous primers, this may be responsible too. Is the region you are amplifying conserved? If not that may be a reason and so you may have to amplify conserved regions in what you are looking for, they are not variable.

hi, i suggest that sequence of your primer might be complementary to certain regions in DNA, thereby primer is binding to regions other than your desired gene resulting in non-specific amplification.

you may reduce non specific amplicon by increasing you PCR annealing temperature.

You can see such bunds except specific band on your gel:

traces of isolated genomic DNA in case on it's great amount or high sensitive methods of detection

pseudogenes in case regions for primer annealing still unchanged and conserved

gene isoforms (in case when gene of DNA fragment is polycopy we can find fragments with different lenthes caused by embedding or deletion of introns, any mobile elements or some nucleotides)

chimera product fragments

unspecific fragments

primers and primer dimers

contamination by another DNA

contamination by restriction enzymes can cause a lot of bands on your gel too

Also note than some dyes especially in case when they used in higher concentrations than recommended (i.e. ethidium bromide) can cause background pigmentation the gel.

Before to choose what to do, can you precise the intensity of the unexpected bands compare to the expected bands?

Also, have you a multiband profile (expected+unexpcted bands) or just monoband profile with unexpected bands?

Go for gradient pcr standardize your annealing also use diluted dna sample and filter tips

It could be many reason. One major reason could be a primer dimer where two primer molecules joined or your primer isn't specific it may amplify two different DNA

molecules at the same time. It could also be the marker may be mixed with a PCR product during loading on to the gel.... Good luck Mohamed

Dear mohamad

all of these explanations are perfect, but I think Tm and Mg concentration are very very important for additional bands. so plz be exact them.

Please also look for any repeat regions in your area of interest. Anyway having some bands is better than not having any. Incorporate changes one or two at time , like may be reducing primers ans cycles or annealing temp, or time,so that you know where to standardize. Also you may try hotstart method.

Check the purity of your DNA preparation, concentration of the primer and finally the buffer.

as Oleg Laptenko mentioned, non specific Banding can arise from nonspecific priming and extension, in this case you can check the primers and be sure about their related specificity. if it is not good, change the primer sequences. sometimes primers are Ok and it is only necessary to change the temperature or timing specially at annealing phase of PCR(based on non specific band is smaller or larger than your desired band, different strategies are required.

finally, be aware about the template or enzyme concentration( or other reagents), more than necessary can affect your PCR data.

hope to hear you about a nice optimization and good results

goodluck

Optimization of your PCR conditions will solve the problem. If your current Tm = X, set a gradient +/- 5 of X, run a PCR and select Tm which is specific to your species. You know that you current Tm is not specific. Good luck Mohamed

The potential causes seem to have been well covered! So, my suggestions to remove non-specific bands would be: check your annealing time, do a gradient PCR to check the melting temperature, reduce primer concentration to reduce primer dimer formation, use hot-start Taq to reduce non-specificity, use less PCR cycles, check your primers as previously explained by others, use a good quality grade Taq. Hope this helps!

Mohamed,

If you use a Master mix (2X), you will reduce the error by half.

Good luck

Hi,

If your non-specific band is bigger than your product it might also be the template DNA. This you can remove by DpnI digestion.

For the rest see other comments.

Ciao,

Istvan