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

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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 ;)

-almost a doctor-


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/無 dissolve in (痞): 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 然. So i took 25痞 from stock dilution and combined with 975 H2O -> so i assume that i have my working concentration of 2,5 然.

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

Additional primer info:

5.2 OD
27,3 nmol
MW 5083


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

they dont give you the Tm? i guess u need to change the annealing temperature 1-2C higher than what u was used now...smearing mean the the pcr amplifying but unspecific bind...10uM primer concentration is good working conc...


Thank for your all replies!

Seems, the problem is solved. Yes, they give me Tm. It says 55 C (on data sheet). Don't trust it!
Tried different temepratures with thermogradient PCR. And wow :) The annealing step should have done at 56C.

Besides, i received the same primer some time ago - and i was given instructions to set annealing temperature to 50C. It worked! But then, when i bought THE SAME primer, the things went wrong.

In general, yes, it was the annealing temperature.

Thanks for your contribution


Yes, Metabion calculates the Tm with a different method than primer3 which I use. I don't look at it either, Ta -4 degs from primer3 Tm works for me.


Trof on Mon Jun 27 11:03:31 2011 said:

Yes, Metabion calculates the Tm with a different method than primer3 which I use. I don't look at it either, Ta -4 degs from primer3 Tm works for me.

thanks for your reply. But the interesting thing is, that the Tm (given on data sheet) was lower than optimal annealing temperature! In other words, the annealin temperature was higher than Tm more than 4C it is important to keep that in mind, that negative PCRs could be related to false Tm temperatures given on data sheet.. Annealing temperature optimisation will be a must for new primers..


I completely and totally ignore anything printed about a Tm on a data sheet. If you do a rational design of your primers, they will almost always work with an annealing temperature of 55C. If they don't, then a gradient from 50 to 65 will find the spot they work at. I have never found the Tm information provided to be of any use WHATSOEVER.

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