Primer dilution --> problem.. - (Jun/17/2011 )

I think your gel is well overloaded. How much of your PCR product are you running? Maybe you could try with less... I've seen this happen with RNA, sometimes what looks like a degraded sample (smear) is actually the result of an overload, easily fixed by running a new gel with lower sample volume.

just my 2 cents

DNR,

Other than overloading of samples, I think there is too much template DNA to start with.
<*>You could try a 5 fold, 10-fold or 100 fold dilution of the template you use for the PCR.
<*> Also, if you total volume is 20 ul, you could also consider lowering the Taq you use to 0.2- 0.3 U.
<*>Because you mentioned, ISSR, I am presuming you are working with plant DNA and if so, you could consider treating your template with RNAse, before proceeding to PCR. It will get rid of smears that you are seeing on the lower end of the gel.

Does DNR, have anything to do with DNR Israel ?

Thanks for your reply.
I ordered primers from metabion. Data sheet says "for 100 pmol/µL dissolve in (µl): 273". So I did that and got, what i call - stock concentration.
So i did another dilution. i've mentioned, that i need working concentration to be 2,5 µM. So i took 25µl from stock dilution and combined with 975 H2O -> so i assume that i have my working concentration of 2,5 µM.

For PCR applications for 20µL reaction mix i use 6,4 µL from working stock - so i get 0,8µM ?

Additional primer info:

5.2 OD
27,3 nmol
MW 5083

thanks

P.S. I'm asking you this, because I ordered new primers and I get smear all over the lanes. Also changed polymerase. And I'm using new dNTP mix. Before that i had positive result using old primer (which is, in fact, the same)by ISSR tech