- 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.
- 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.
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 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.
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
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.
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!
There are several reason for your problem, as you said those bands were far from your target band those might be primer dimer. but if that is primer dimer the band should be smaller than your target band. you can overcome this problem by hot start.
or your primer is not specific to your target region if that is the case, you can try touchdown PCR that helps to specific binding.
Be careful with contamination because different organism have same sequences with only few nucleotide changes, if you contaminate you get more than one band.