Hi Tomasz,

yes contamination is primer specific...the wrong primers will not anneal or amplify.. Pcr product only self anneals when it is present in huge amounts which only happens when amplification is happening so with the right primers.. Contamination does not have to be in the primers ( which should be dissolved in TE not water ideally) but if it gets into any part of the pcr reaction....reagents, pipettes, tubes,dust in the working area , gloves that have been dipped in gel running buffer after a gel has run then it will amplify better than your dna and take over the reaction even in the negative control which is not really negative if it is contaminated. When product self anneals it may not anneal perfectly but at the low primer annealing temperature and with lots of product one part of the forward strand may anneal with a different part of the reverse strand and produces a longer product as the 2 strands have annealed asymetrically and with more cycles this only gets worse.

Concerning re amplification this works well with nested primers but if you are re amplifying pcr product it is best to dilute 1:100 and amplify for 10-20 cycles or over amplification and smearing will become a problem.

I think that it is post pcr contamination of pcr product. Bearing in mind every 10 cycles is 1000 times more product even a tiny contamination takes over and becomes so numerous that it self anneals and you end up with smears of all sizes. Ethanol washing is no good as ethanol just dries dna not removes it.. Order new everything and set up a run in another work area/lab with someone elses pipettes and tubes and pcr machine . If this pcr is clean start a major clean up of your working area with bleach and much disposal of reagents

Remembering each 10 cycles is 1000 times more item even a small tainting assumes control and turns out to be numerous to the point that it self toughens and you wind up with smears of all sizes. Ethanol washing is no great as ethanol just dries dna not evacuates it.. Request new everything and set up a keep running in another work region/lab with somebody elses pipettes and tubes and pcr machine . In the event that this pcr is new beginning a noteworthy tidy up of your working territory with blanch and much transfer of reagents

regards

Thank you very much for all your answers.

Paul are you suggesting that this contamination is specific for those particular primers, I mean that this pair anneal to it and the other pair doesn't? If it self anneals shouldn't it be unspecific to the reaction and appear in all reactions? I recently ordered brand new primer batch with the same sequence and dissolved them in ultra pure water and still have this smear. If that would be contamination with pcr product of this particular reaction shouldn't it produce just a sharp band rather then a smear? I did in the past pcrs using pcr products as a template for this very reason.

Rachel I've tried different water kinds starting with just MilliQ through pure water from a commercial kit and ending with commercial ultrapure. My last master stock I prepared with ultrapure water and still with no success. If this is some sort of contamination I can't understand why smears are not the same size and in all samples. It strikes me as weird. Especially on the picture named "smears" in 2 first lanes after DNA ladder two negative controls are not the same. I'm using filter tips for a while so I would say that aerosol contamination would be unlikely but I will try with someone else's pipettes.

Hi Tomasz,

yes contamination is primer specific...the wrong primers will not anneal or amplify.. Pcr product only self anneals when it is present in huge amounts which only happens when amplification is happening so with the right primers.. Contamination does not have to be in the primers ( which should be dissolved in TE not water ideally) but if it gets into any part of the pcr reaction....reagents, pipettes, tubes,dust in the working area , gloves that have been dipped in gel running buffer after a gel has run then it will amplify better than your dna and take over the reaction even in the negative control which is not really negative if it is contaminated. When product self anneals it may not anneal perfectly but at the low primer annealing temperature and with lots of product one part of the forward strand may anneal with a different part of the reverse strand and produces a longer product as the 2 strands have annealed asymetrically and with more cycles this only gets worse.

Concerning re amplification this works well with nested primers but if you are re amplifying pcr product it is best to dilute 1:100 and amplify for 10-20 cycles or over amplification and smearing will become a problem.

Thank you Artur for your answer.

I'm using 40 cycles right now because with 30 or 35 I didn't get the amount of pcr product I need for downstream analysis (yes I could increase volume of the reaction but for optimization purposes I was conserving reagents). I thought that's because I'm using genomic DNA as a template and increasing cycle number will give me the amount I was getting using plasmid or purified pcr product as a template.

For now I resolved this problem by ordering new set of primers with different sequence targeting the same region.

Thank you all for your input.

what's yours annealing time?