If your non-specific band is at 800bp and your band of interest is at 400bp, why not just decrease the extension time?  Typically protocols call for 1kb for 1 min.  So for a 400 bp product decrease extension to 30 sec.  Then you should definitely not see a band at 800bp.

good luck! 

Non specific bands in my opinion can be reduced by altering temperature.. try to change annealing or denaturation renaturation temp and its time.. or try to run a gradient for similar (temp as well as time interval of temp) removal of non specific bands..

Hope this helps,

Best wishes,

Tejas

Maybe try a Hot Start PCR... It reduced my unspecific bands when using degenerated primers....

Hi guys,

Thanks for the suggestions. I have just looked up the range of PCR master mix I use and there is a hot-start version I can buy so I might give this a try!

"Touchdown PCR" (or decreasing annealing temperatures from a higher temperature down to a lower temperature by a set amount each cycle) can help increase specificity.

See "touchdown PCR" here http://cshprotocols.cshlp.org/content/2009/4/pdb.ip66.full

guiede in publishing paper

Is the short fragment amplified to be used for DGGE? Does the primer have long GC chain?

That can be the problem. I have experienced something similar. As suggested above, try to do a Tannealing gradient and if you still get unespecific band, go for the touchdown PCR.

the concentration of Mg can also make a difference. In some cases I have also used DMSO in the Master Mix to increase the specificity of the reaction.

Dear,

just use 30 cycles and increase annealing temperature, hopefully its work if not then go for gradient PCR with higher annealing temperature> be happy

Try to increase 3 or 4 degrees the annealing temperature. Low annealing temperatureS use to be the main cause of unspecific bands. If you have no success with this, then I would try modifying the Mg concentration or adding DMSO to the cocktail. But I think it will be enough increasing temperatures.

Hi Emma,

You can try with step up or step down PCR. These kind of thermal programme help to bind the primers to their specific binding region in first few stages (eg first 5 stages) and then the second (which is higher in case of step-up and lower in case of step-down) annealing temperature for remaining cycles (eg 20) help you to amplify those specific bound region.

or you can try with adding some BSA. If your sample is environmental sample then add 0.25 microlitre of 10mg/ml BSA and if the samples are from semi-pure/pure culture then add the same volume of 1/2mg/ml BSA for 25 microlitre total PCR reaction volume.

Hope it will help you. Best of Luck !!

Dear Emmah Have you had any success to slve your problem? I have the same problem now!

Hi Mahdi,

I found changing the batch of PCR master mix and not PCR'ing above 30 cycles with the PCR program I was using (which had quite long extension times) as the template seemed to amplify well solved any issues.

Dear Bennett,

I want to use Touch down PCR program for amplification. But I don't how to set the condition in PCR machine. I know about the general PCR condition setting. But I do not have any idea about decreasing temperature per cycle. Could you please help me to sort out this problem.

Regards,

Sailesh

If your non-specific band is at 800bp and your band of interest is at 400bp, why not just decrease the extension time?  Typically protocols call for 1kb for 1 min.  So for a 400 bp product decrease extension to 30 sec.  Then you should definitely not see a band at 800bp.

good luck! 

I concur with Sarah reducing extension time should work though I must say your extension time seems too long I am doing 16S rDNA PCR with an extension time of 30s on a 1.5 kb amplicon and its working just fine.

I got same problem too, my target size is 1,000 bp but always show in 3,000 bp. I had already manipulate the temperature of annealing. the primer ™ is about 52 degree. but I still get non specific band and still low band in target size.

my friend told me that the problem maybe in exon junction between exon 1 and exon 2, then I try to check it.

someone have some advice for me? thanks

hi

you should decrease annealing temprature and remove mgcl2 from master mix

I am facing same problem with my gene too, i am going to amplify 471bp but instead of 471 i am getting about 800bp, i try so many tips as mention above, any thing else please suggest!

Hello friends!! I need you help. I´m trying ton amply a gene AATS1 in Diptera. According to the protocol than I based on. Im using a TOUCHDOWN PCR. Actually Im using GREEN GO TAQ MASTER MIX, and the first time that I proof, I didnt get a good band. So I modifyed the tempertures from the first and second face of my PCR. Fortunately I got my specific product, (591pb) but I couldnt removed a non specific band of (200pb). If you have any information please tell me.

Hi Maria,

You could try modifying the temperatures of the PCRs further to achieve the correct balance between obtaining the correct size band and avoid the non specific amplification - the higher the temperature the more specific you will be.

You could try using a hot start master mix? This is designed to reduce non-specific amplification.

Also, you could try different PCR master mixes to the one you are using and see if that makes any difference to the results.

If you cannot remove the non-specific band, you can gel extract the band of the correct size instead using a kit, but this takes more time and isn't the most efficient process. It depends on how many samples you are processing with the PCR in question.

Maria, nested PCR is the easiest way, probably, if you need PCR fragment for sequencing/cloning. 200 bp fragment can be a part of larger one (due to false annealing site within the target fragment), but it can have only partial homology and it will spoil your efforts to sequence the target gene.

I am facing reverse of it too. My band size is 119bp and I am getting 50bp band in my negative control.

Lee, you have to accept that  primers or water in negative control was contaminated by alien DNA.

The pcr was showing prime dimers. I added 5% DMSO. The prime dimmers disappeared, however appeared a nonspecific band of 150bp. Can DMSO cause this?

Non-specific bands not always undesirable or false negative results it may have correlation with upper and lower generations , But you could decreases the concentration of DNA (25-100 ng/ul) and primer (10 pmol) or using a different primer on a different locations in the genome you should use a primer with high specificity,

My regards.

If you exhausted all the ways and still get non-specific bands. You can try purifying your target gene through gel extraction then run PCR again using your purified gene as your template.

Was facing the same problem. Double check your primer sequence and try online PCR tool first. Than optimize annealing temperature and time as well as extension time too.

I expeience a similar problem, my target band is about 1,6 Kb that I get with a non specific band of about 800 bp; Is there anyone that could help me?

Reduce MgCl2 concentration and primers also. Set gradient PCR increasing annealing temperature.