For SYBR Green qPCR it is best to use primer concentrations that do not entice primer dimer or other non-specific amplifications to occur. Anywhere from 100 nM to 600 nM of each primer (in the final reactions) has been shown to be the most efficacious range to stay within in the vast majority of situations. Typically, to hone in on the best concentrations of forward and reverse primers for an assay, a matrix is run from 50 nM to 900 nM primer concentrations with forward and reverse primers in different amounts across the matrix and in the same amounts as well across the matrix.  Asymmetric qPCR (fwd and rev primers at different concentrations) sometimes proves to be more efficacious than symmetric qPCR.  One can only discover this empirically by running a matrix.

The vast majority of commercially-designed assays for SYBR Green qPCR typically use between 300 and 600 nM of each of the fwd and rev primers in final rxns. Often 1 uM primer concentrations entice too much primer dimer formation - but, can work perfectly and robustly in optimal situations. The more primer one can use (without dimer and non-specific side reactions happening) the better, as it is like turning up the volume knob on a stereo to get more signal - and, in agreement with António Maximiano Fernandes: you probably never want to exceed 1 uM primer concentrations as it is near over-kill at 1 uM and above...

For primers concentration in qPCR is 1 uM

To determine the best concentration dont exced 1uM

In qPCR most critical is dna template concentration

For SYBR Green qPCR it is best to use primer concentrations that do not entice primer dimer or other non-specific amplifications to occur. Anywhere from 100 nM to 600 nM of each primer (in the final reactions) has been shown to be the most efficacious range to stay within in the vast majority of situations. Typically, to hone in on the best concentrations of forward and reverse primers for an assay, a matrix is run from 50 nM to 900 nM primer concentrations with forward and reverse primers in different amounts across the matrix and in the same amounts as well across the matrix.  Asymmetric qPCR (fwd and rev primers at different concentrations) sometimes proves to be more efficacious than symmetric qPCR.  One can only discover this empirically by running a matrix.

The vast majority of commercially-designed assays for SYBR Green qPCR typically use between 300 and 600 nM of each of the fwd and rev primers in final rxns. Often 1 uM primer concentrations entice too much primer dimer formation - but, can work perfectly and robustly in optimal situations. The more primer one can use (without dimer and non-specific side reactions happening) the better, as it is like turning up the volume knob on a stereo to get more signal - and, in agreement with António Maximiano Fernandes: you probably never want to exceed 1 uM primer concentrations as it is near over-kill at 1 uM and above...

1uM of each primer??You are rich guys)))

5-10 pmol/ per reaction of each primer is enough for Sybr. Much depends on your Real Time instrument, but sybr is very sensitive. For greater primer concetrations, you shall need more Sybr, but in greater ammounts it inhibits PCR.

Good luck

Hi Andrey,

Just to repeat a portion of my post from above:  "For SYBR Green qPCR it is best to use primer concentrations that do not entice primer dimer or other non-specific amplifications to occur. Anywhere from 100 nM to 600 nM of each primer (in the final reactions) has been shown to be the most efficacious range to stay within in the vast majority of situations."

Primers are actually not expensive in general, however, dual-labelled probes are. Rich? Don't we all wish for more funding - especially in science these days ;]

Also, if one is running 10 uL-size qPCR reactions, 5 to 10 pmol per reaction is 0.5 uM and 1 uM primer concentrations, respectively.  But in 20 uL reactions, it would be 0.25 and 0.5 uM primer concentrations.  Sometimes the numbers confound, astound and surround. And, agreed, some very good SYBR Green qPCReactions can indeed run perfectly and robustly at 100 nM (0.1 uM) primer concentrations. 

Empirical testing of a matrix of primer concentrations often helps.

Jack,

uM is not a concentration. This is a measure of ammount, that is missed by many)) Our final concentration in reaction is (5pmol/mcl) per reaction (1mcl of primer in 10ul reaction volume). In total ammount per ml, it is 5nM. In fact my order of primers never exceeds 0,02uM. Maybe somewhere you have found the cheep source of synthesis, but the common price for 1uM of purified pare primers is $250. This is extremly expensive))) Nevertheless I wish you, as well as to most of us to have as much financial support in science, as possible.

Andrey,

The definition of Molarity disagrees with you.

see:

https://www.thoughtco.com/molarity-definition-in-chemistry-606376

Any expression of Molarity is a concentration (by definition).

Molarity = (g/MW)/Liter = moles/Liter = M (Molar) concentration

moles of something per Liter = Molar concentration

mmoles of something/Liter = mM (millimolar) concentration

and pmoles/L = pM (picomolar) concentration

and pmoles/mL = nM (nanomolar) concentration

and pmoles/uL = uM (micromolar) concentration

and so on...

Frequently primers are received as lyophilates (e.g., a certain amount of picomoles or nanomoles) to which one adds a certain amount of buffer or nuclease-free water or TE pH 8.0 to in order achieve a desired stock primer concentration.

In your example, if you are achieving a 5 nM concentration of a certain single primer in a 10 uL rxn by adding 1 uL of primer, that means your starting primer stock concentration was 50 nM.

When you state "Our final concentration in reaction is 5pmol/mcl)" you are saying that your final primer concentration is 5 uM since 5 pmoles/uL = 5 uM.  (You probably meant to say 5 pmol/mL, since 5 pmol/mL = 5 nM).

5 nM concentration of primer in a qPCR reaction is very low, normally one would use nearer 500 nM instead.  Perhaps your stock concentration is actually 5 uM and, after adding 1 ul of that to a 10 uL qPCReaction, you are achieving 500 nM final primer concentration.

$250 for 1 umoles (micromoles) of primer (if that's what you meant above) is actually a very bad price. Too bad it costs that much for you.

1 umole of primer costs $2.10 per base at IDT (Integrated DNA Technologies) - so a 25 base primer would be about $52.50.  Better pricing from IDT.

Attached here is a tool one can use for calculating precise primer dilutions using primer molecular weights and extinction coefficients of each primer in conjunction with spectrophotometry.