Please see the following factors that can contribute to unspecific, smeared PCR products, and suggestions how to avoid it:
too much starting template
Check the concentration of the starting template. Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using these serial dilutions.
carry-over contamination
If the negative-control PCR (without template DNA) shows a PCR product or a smear, exchange all reagents. Use disposable pipet tips containing hydrophobic filters to minimize cross-contamination. Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis.
enzyme concentration too high
When using HotStarTaq or Taq DNA Polymerase, use 2.5 units per 100 µl reaction.
too many PCR cycles
Reduce the number of cycles in steps of 3 cycles.
Mg2+ concentration not optimal
Primer concentration not optimal or primers degraded
Repeat the PCR with different primer concentrations from 0.1–0.5 µM of each primer (in 0.1 µM steps). In particular, when performing highly sensitive PCR, check for possible degradation of the primers on a denaturing polyacrylamide gel.
Please see the following factors that can contribute to unspecific, smeared PCR products, and suggestions how to avoid it:
too much starting template
Check the concentration of the starting template. Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using these serial dilutions.
carry-over contamination
If the negative-control PCR (without template DNA) shows a PCR product or a smear, exchange all reagents. Use disposable pipet tips containing hydrophobic filters to minimize cross-contamination. Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis.
enzyme concentration too high
When using HotStarTaq or Taq DNA Polymerase, use 2.5 units per 100 µl reaction.
too many PCR cycles
Reduce the number of cycles in steps of 3 cycles.
Mg2+ concentration not optimal
Primer concentration not optimal or primers degraded
Repeat the PCR with different primer concentrations from 0.1–0.5 µM of each primer (in 0.1 µM steps). In particular, when performing highly sensitive PCR, check for possible degradation of the primers on a denaturing polyacrylamide gel.
in addition to the excellent suggestions above try a gradient of annealing temperatures as a hotter annealing temperature may imptove your amplifications. you may be annealing too cold so the primers can stick in many places on the genome.Check the primer sequences particularly to make sure that there is not homology with any repeat sequences. these are very common and exist in both orientations so even one primer can amplify between 2 repeats. You also seem to have a primer dimer problem so using a hot start enzyme and less primer may help this
Can you tell me what type of template you are using? I am assuming genomic DNA? if it is, sometimes smearing occurs due to DNA degradation. Make sure to use DNase free water. Since you are seeing smear in your negative control too, may be your water is contaminated?
if negative control also giving smear result, maybe one of your mixture contanimated. try to prepare a new primer mix. Aliquote all your mix (MgCl, neulotides, primer, buffer, etc) into tube.
All above mentioned suggestions are good and I also think such types of problem you have. Negative control is very important to identify DNAse enzyme contamination in your water, check it first, then try other solution.
Troubleshoot contamination by including a negative and positive control respectively in your extraction...then run your PCR using your extracted controls and a set of controls that worked in the past (this should not be re-extracted)...If smears appear on the lanes for freshly extracted controls and doesn't appear on the lanes for the set of controls that were not re-extracted, then the contaminant is linked to he extraction process...if both sets of controls show smears, then the master mix is the source of possible contamination.
The above suggestions are right, so try to run your gel with low voltage, with a water control. if smears still appears on control then the suggestion about contamination is right if not, try to run your extracted samples again before PCR to ensure you have enough DNA in your samples
You have used high concentrated DNA for this, you are observing such smears. So, lower the amount of DNA or dilute it and used below 500 ng/ul per reaction as possible.Best to keep DNA concentration in between 25-100 ng/ul per reaction, what i have watched in most of the kits or master mix preparations.
Better to run you sample's genomic DNA in 1% agarose gel with 1X TAE buffer for 15 minute to check the consistency and quality of your genomic DNA.
If your sample DNA's contain much protein like when the ratio of 260/280 is less than 1.6 or less then that. Then the amount increase in DNA per reaction with inhibit the pcr to amplify due to extra load in protein.:-(
Paul Rutland Sir, well said about annealing temperature and gradient pcr. So, you can do a gradient pcr according to your primers Tm temperature to find a better annealing temperature that yield much pcr product. I myself always apply gradient pcr in every newly designed assay to find a perfect annealing to increase the yield .
Most important things you said Smearing in"Negative control". So, may be one of your component in pcr reaction is contaminated and if your gel electrophoresis chamber contain old buffer then you must change it because this also can causes smearing in Negative control, where you didn't add any tamplet DNA.