HI Jebelli,

There are many causes of primer dimers, in this case it could be that your samples where you are seeing problems have slightly higher salt concentration. One of the easiest ways that I have found of  overcoming this is to raise the annealing temperature by a few degrees.

you have contamination of post PCR primer dimer caused in tthe same way as pcr product contamination. You will need to clean everythinh in your working area with bleach ans thoroughly wash down the area. Try settinh the pcr up in another lab or at least another area of your lab away from any area that has been use dfor your pcr or checker gel running. Pipettes will also need cleaning or borrow some. Run plenty of negative controls. If the controls are positive then reagents or laboratory surfaces are contaminated

Hi dear Steve

Thanks your comment. I have this problem for one of my genes (for example gene A). these samples had so good results for other genes (B, C etc.). in addition, when I repeated samples that I had good results for gene A, I found primer dimer as well as main pick. I increased annealing tem. but results didn`t change.

Hi dear Paul.

Thanks your suggestions. But I use this area, machine, pipettes and other materials for other genes and I don`t have any problem with those genes.

Hi Jebeiili,

Have you tried a new batch of primers, it is possible that your primers have gone off, although they are robust they do have a finite life. I our lab we tend to create two or three forward and reverse primers, as they are relatively inexpensive, and test them against each other. If there is a problem with one set in the future, it means that we have others to fall back on as a check.

The other thing is the PCR block, I do not know how old you machine is, but with older blocks you can start to get failure with specific positions, which is easier to spot with plates, but is less obvious with tubes, have you noticed a trend?

hi Jebelli,

contamination either of primer dimer or post pcr product is absolutely primer specific . . You can add one amplimer in any quantity to other primers and they will not work .You are just unlucky to have contamination with this primer set. You can get away from contamination of post pcr material by using one or 2 new primers outside of the amplimer or , in this case, just design different primers. There is some aspect of your technique which allows contamination such as running checker gels in the pcr setup area or opening tubes/plates too violently causing aerosols  which you will need to think about or it could happen with other primer sets in the future

Hi, you may try designing new set of primers for this particular gene, did you check if the existing once have more complementary bases which could allow self binding of the primers to an extent that might give a second smaller peak.