primer dimer and non specific bands

[rair]

I'm doing nested pcr using Bioron mastermix beads 50μl reaction and according to the protocol I follow the first round primers are used in 0.2μM concentration,The second round primers are in 2μM conc.Not only primer dimer bands of 50bps appear with all samples but other bands appear at about 100bps but not with all samples .My target gene is 322bp which appeared once.could primer dimer prevent target amplification in other samples and what are those non specific bands.thanks

[SebaQ]

Try checking the sequence of the primers for dimers, rise the annealing temperature to make the pcr more specific and if you still have problems, try to just excise from the gel the band you want to work with.

And yes, primer dimers could prevent target amplification because the primers are "consumed" by generating dimers and then they won't be useful for the polymerase

[rarhong]

I've checked my primer with DNA calculator previously, the result was none primer dimer. But when I use it for PCR at the concentration of 20microM there are always none specific bands. After I changed the concentration to 1microM (based on the TaqPol protocol) unspecific band dissapears. I think the final concentration of the primer in the PCR mix also rules..

[caroline]

1) i performed a pcr using two different primers on mushroom fruitbody and sclerotium of one of the mushrooms. the genus specific primer designed gave one band for all samples but the ITS 1 (forward) and ITS 4 (reverse) gave one band for fruitbody and two bands for sclerotium (one faint band on top of a major band). what is the cause?

2) How do i use this result to identify my samples?