It use to be that 1600bp was a standard size for PCR, but by today's standards, that is a big size for an amplicon. Firstly, we need more information about your cycling conditions. At 1600bp, I would say that your elongation time should be at least 90 seconds (usually 1Kb/min is a good rule). Are the "non-specific" bands smaller than your desired amplicon? You could also try linearising the vector. It is quite rare that amplification from a cloned vector produces non-specific bands. I would say diminish the concentration of your template; remember that unlike amplifying from genomic DNA, when amplifying from a vector, it is ALL target. You really need very very little template.

You should use some additives like betain and BSA. Also trying the gold Taq or any other efficient taq.

It use to be that 1600bp was a standard size for PCR, but by today's standards, that is a big size for an amplicon. Firstly, we need more information about your cycling conditions. At 1600bp, I would say that your elongation time should be at least 90 seconds (usually 1Kb/min is a good rule). Are the "non-specific" bands smaller than your desired amplicon? You could also try linearising the vector. It is quite rare that amplification from a cloned vector produces non-specific bands. I would say diminish the concentration of your template; remember that unlike amplifying from genomic DNA, when amplifying from a vector, it is ALL target. You really need very very little template.

Are the non specific bands primer dimers? Or longer products?

If your current PCR reagents work with other targets/primers, I would not recommend to change them.

If you don't see a difference in temperature gradient, maybe you should change the temperature range. Apart from this, you may increase amplification time, optimize primer concentrations, add a few more cycles.

Based on the information you provide I'd say you have already played around with PCR parameters. In that case, if you must absolutely have a clean, specific PCR, I think your best bet is to redesign the primers.

If that is not possible, and assuming your reagents work and your PCR setup is OK, can't you just gel-purify your band and do away with PCR optimization? If not, I would try a touch-down PCR, or a multifactorial experiment varying template concentration, Mg++ concentration and annealing temperature (easy if you have access to a gradient thermocycler). You can also play with some additives, as suggested by Raghvendra Singh above. Betaine is great, and there is also DMSO. Google around for specifics.

Another possibility is to run the result of your PCR on a gel and then, using a plastic toothpick, stab your band, rinse it into a few microliters of water, and use that as template for a new PCR. This will only work if the non-specific amplification is caused by the primers binding outside your target gene; it won't rid you of primer dimers, etc.

Dear friend, I suggest you do a contamination check for your primer and reagents. Sometimes these things can happen because of contamination. Just run a PCR without template and lets see what happens.

Best of Luck....

Thanks from all of you,elongation time is100second.non specific band are smaller,i dilute my template five time but i don't see specific band,altough non specific are present.i am sure about primers.

If you have non specific bands try touch down PCR. double check your PCR conditions. as Alejandro Martin mentioned Mg concentration is an important factor. Read Mark Laflamme notes again, it is perfect.

Use high fidelity phusion polymerase rather than using Taq and Pfu polymerases.

phusion polymerase will amplfy only yours desirable amplicon.

in most cases it is reliable and efficient.

Non-specific bands with a cloned vector as template are really quite rare as Mark Laflamme has written. Maybe the concentration of your template is too high - using a vector as template I would recommend to dilute it at least 1:100.

Check your Primers, especially for potential formation of dimers and hairpin loops and for Tm difference, if necessary optimize them.

My advice for you is to use T7 DNA polymerase or sth similar to it.

T7 DNA polymerase is highly processive DNA polymerase allowing continuous synthesis of long stretches of DNA.

Add an extra five cycles

Which polymerase do you use? I was tearing my hair out trying to optimise a PCR with Pfu, using Pfu Ultra, it works just great.

Adrian@Alice@Mohamed Mahdy thanks.i do my reaction in 20 micro volume(1mic dntp5mM,1mic mgcl2 25mM,my template is plasmid contain desire gene1614bp and enzyme that i use is taq DNA polymerase) my program in thermocycler is(95,94,62.5,72,72)degree,but now i use DMSO and get band:) anyway thanks.

I was having a similar problem, and it turned out that all through grad school we had hot start taq, while the lab I am doing my postdoc in does not.  Luckily, this is really easy to fix- if you are currently setting up reactions on the bench and starting your pcrs once all the tubes are in the machine, stop doing this.  Set-up mater mix/tubes/etc on ice and place immediately into a pre-heated PCR machine when complete.  Regular taq can make all kinds of crazy unexpected things if left unattended at room temperature....

There are many reasons for light bands after electrophoresis of PCR products, some of them summarised as follows:

1)The gell concentration was high may be more than 0.8. 2) The polymerization of the agarose gel incompletely. 3) The DNA samples are old preparation. 4) The staining with ethidium bromide was not complete, with less stain or bad stain. 5) The DNA fragment was small. 6) The electic field was unsuitable 7) The steps or cycles of PCR are not good. 8) The running buffer was not enough water to flood the gel. 9) the field of the running tray was not suitable. 10) The volumes of DNA and primer which added to the PCR reaction was unsuitable. 11) the gel was stained for an old period. and more.........