Hi Devi Prasad, I have tried the same, doing re-amplification with the same primers and also got smear. However, when I use nested primers, the results are OK.  It is true that on the gel (after the first PCR) you see only your expected amplicon. However, be sure that other DNA-fragments are there in your tube which are (almost) invisible on agarose gel after the first amplification but start to appear after a second run of amplification (unfinished amplicons, undesired amplicons, genomic DNA fragments, primerdimers etc) resulting in smear. Nested primers causes a selection of amplification of the desired amplicon, leaving aside (all or most of) the other unwanted fragments. So, if you want to use the same primers on the amplicon, you should get rid of all these unwanted products first. To achieve this, I do the following: I put all of my amplicon after the first PCR on agarose gel and cut out the band of interest. Out of this piece of agarose, the amplicon is purified (for example: Wizard SV Gel and PCR Clean-Up System from Promega). The purified amplicon is than used in a second amplification run using the same primers. Hope this works for you too.  Best regards.

Hight concentration of template and enzyme(polymerase) in the PCR reaction can cause smearing.

so dilute your template and use fresh enzyme (.5-.3 ul/rxn)

Increase Annealing Temperature to 59 or use touch down PCR, Start your PCR with higher annealing temperature, lets say ( I assume your are using 57 annealing temperature) 62 and decrease 1 degree per cycle and go down to 57 and then run normal PCR cycle (30 or 35) that you a  using in routine PCR. for further details if you interested please follow the link below

http://www.nature.com/nprot/journal/v3/n9/full/nprot.2008.133.html

Yes. Had the same problem. Reduce all of these parameters: Amount of template, amount of enzyme and the number of cycles. You could add about 0.5 uL of DMSO to a 25 uL reaction so as to decrease non-specificity. It worked for me! Try it and let me know.

Thanks for the suggestions. The first run (from gDNA or cDNA) gives a single band; no non-specific bands or a smear. I had tried 1:1000 dilution of the template (i.e., taking the PCR back by about 10 cycles) and still got the same. How much more is it recommended to dilute the template?

I shall try reducing the enzyme and cycles, but I did not understand why, though.

I suggest you to get rid of primer dimers in your PCR product by using MicroCLEAN (http://webscientific.co.uk/microclean-dna-cleanup-reagent-10-x-1ml.html) before using it as template for next PCR,

Best of Luck

Discussed with a friend who works with RNA and expression profiling as well. If smear persists, then something is getting degraded. Did you include a control? I mean to say, you should have done cDNA synthesis for a constitutively expressed gene as well ( like GAPDH in plants). Then you should have used the GAPDH cDNA mix as template and run a PCR paralelly. If you had done that and still facing the problem, it means contamination or degradation or excess of ions. And one more thing... how specific are your primers? Validate the primers if they have 18-24 nt, GC content 40-60%, Tm(salt) not more than 60 C and if the DeltaG for a primer dimer/ secondary structure is less.Hope this helped.

@deepak, The first time I run the PCR, there is no problem at all. Single amplicon ~200nt. The primers I use are meant for qPCR and they do well otherwise, but for this issue. And no bias; I have got this for multiple genes. For something to get degraded, the starting material has to be larger, right? But the conditions I provide can amplify only about 750nt (ignoring the final 10 min extension) and the smear is from 200-2000nt.

@artur, As suggested, I'll try lower templates and cycles.

@arshad, I had once cleaned up the product after I got a smear. I could try it the other way.

The reasons that a smear on a gel is obtained after staining for protein or nucleic acids, whichever the case may be, or on the autoradiograph after X-Ray film exposure, are multiple ones.  Whenever something does not work you have as many possible explanations as you may think.  However, I would first check for the ionic strength of the application buffer.  Otherwise, I would check with the PI of the project who got funded for the project.  If the latter does not work you may be in the wrong lab, even if there is funding for the project.

Hi Devi Prasad, I have tried the same, doing re-amplification with the same primers and also got smear. However, when I use nested primers, the results are OK.  It is true that on the gel (after the first PCR) you see only your expected amplicon. However, be sure that other DNA-fragments are there in your tube which are (almost) invisible on agarose gel after the first amplification but start to appear after a second run of amplification (unfinished amplicons, undesired amplicons, genomic DNA fragments, primerdimers etc) resulting in smear. Nested primers causes a selection of amplification of the desired amplicon, leaving aside (all or most of) the other unwanted fragments. So, if you want to use the same primers on the amplicon, you should get rid of all these unwanted products first. To achieve this, I do the following: I put all of my amplicon after the first PCR on agarose gel and cut out the band of interest. Out of this piece of agarose, the amplicon is purified (for example: Wizard SV Gel and PCR Clean-Up System from Promega). The purified amplicon is than used in a second amplification run using the same primers. Hope this works for you too.  Best regards.

Is there a specific reason why you are re-amplifying from a PCR product? Is this just to obtain more product for further work. If so, don't think you specifically need a nested PCR for that purpose because your amplification yields a single specific band. I would advice cloning your insert into a plasmid and using the plasmid as your PCR template. That way your insert is more stable and you can make glycerol stocks for long-term storage and future experiements.

Maybe try this:

1. You mentioned that your first PCR product is of the expected size. Purify that band from a gel using a gel purification kit. You can even run a small 2-5ul aliquot on a second gel post-purification just to ensure the extraction worked.

2.Clone your insert into a plasmid (TOPO, pGEMT etc) using bacterial cells and perform a plasmid isolation and purification. For validation, perform a restriction digest on a portion of your sample (usually 1ug) using an enzyme that cuts at the boundaries of the insert (eg. EcoRI, depending on the plasmid) and run the digest on a gel for confirmation. For even further confirmation that your insert is an exact match of what you are amplifying, you can send these samples for sequencing. I would definitely recommend sequencing.

3. Use your validated plasmid for your PCR reactions etc. I don't think you will experience smearing since your primers seem to be specific enough in your first reaction.

@Almas, The reason for a reamplification has been different at different times. Once for using as positive control, once for sequencing, once for scaling up, etc. Like some of you have suggested, gel extraction is a good option. But not all of the downstream applications have not demanded the resource. I shall consider doing that.

@Deepak, I tried diluting the amplicon 10^3 and 10^6 times, and with 1 or 0.5 ul of Taq Pol, for 25 cycles. 0.001 dilution gave clear bands with less smear than before; 0.5 ul Taq had more (desired amplicon:smear) ratio compared to 1ul Taq. 0.000001 dilution did not amplify.

i think that most of the problem is the efficiency of pcr. every 10 cycles there is 1000 times more product so that the amount of product is huge and re-annealing with itself becomes more likely than the primers annealing and you get long concatomers of product annealing with itself partially then extending. Nested primers are very good but if you want to re-amplify dilute your product 1ul to 100 ul and amplify 1ul with only 10-15 cycles as Devi suggests

@Artur, I want to give the product for sequencing using the same primers. Though I might not see a similar smear with intermediate dilutions, I cannot rule their presence out. I was also thinking of a shorter extension, probably in addition to a gel extracted product.

oh I see. And for Sanger, we usually get ambiguous reads till 50-100nt. So if I sequence from both ends, I hope to cover the full sequence.

Hi Devi,

I did PCR before and I got same problem. But when I tried to change the annealing temperature I got just one clear band. The reason for getting more than one band that means the primer does not bind the specific region, however; the primer bind all the regions (specific & unspecific regions) that having the same annealing Tm, and when you will try to decrease or increase the annealing Tm you will get just one region or might get more. In this case you will keep trying until you will get one band.   good luck ..........   

Aisha, thanks for the suggestion. But the first time I do a PCR, I get a single clear band. Only when I try to reamplify using that amplicon as the template, I get a smear.

 @Devi Good day to you. I want to ask how were you able to resolve this challenge as I am currently facing similar challenges with my work. 

Please kindly assist. Thanks

@Adebiyi, Hi! I could not exactly resolve the problem. One thing that helped was, diluting the amplicon before using it as a template; 10e3 or even 10e6 fold if necessary.

Thank you dr....I will do just that