Question about Realtime PCR primer concentration.... - (Apr/24/2006 )

Hello

I'd like to ask about primer concentration for Realtime PCR.

In Roche manual, they recommand 0.5uM or 1 uM.

Some guy, he recommand 5pM or 50pM such as classical PCR.

The other paper, they recommand 50-200nM.

Which concentration is correct?

Please, help me...

-xenopuswnt-

there are many ways to do it...you will probably have to optimize for your template

I find that mine works best in the 200-250nM range

-aimikins-

Thank you for your kind reply~

QUOTE (aimikins @ Apr 24 2006, 05:19 PM)

there are many ways to do it...you will probably have to optimize for your template

I find that mine works best in the 200-250nM range

-xenopuswnt-

I've just finished optimizing my primers. I chose to evaluate them at 100, 200 and 500 nM concentration. So...I prepared the various primer dilutions and went with 1 ul of cDNA obtained from 20 ul final volume of cDNA (reverse transcribed from 1 ug total RNA). I found that it does depend upon the primers but 200 nM was usually pretty good for the genes I selected. In two cases the tm looked better at 500 nM so I'll use those for the two specific genes. It's a really good idea to do these experiments. You can also get a rough idea of the amplification efficiency by looking at the log melting curve and seeing the slope of the curve.

Hope this helps!!

-ranabear-

QUOTE (ranabear @ May 1 2006, 02:23 PM)

I found that it does depend upon the primers but 200 nM was usually pretty good for the genes I selected. In two cases the tm looked better at 500 nM so I'll use those for the two specific genes.
Hope this helps!!

500nM is really a lot. What is the volume of your reaction? I work with SYBR Green and take 20pmol of each primer and 0,5 uL of cDNA (obtained from 500ng mRNA). My reaction volume is 20 uL. However the efficiency is below 70% (slope = - 4,4). I don't know why. Melting curves look nice, so my primers are ok. I wonder if I should increase their concentration?

Please help me

-Ewa-

well, I just found out that my NTC give a signal (ct is about 5, while other ct's for my samples are above 15). But on the melting curve for NTC's I don't see any peaks, so there are no primers dimers. What is this?

Maybe I should decrease the concentartion of primers - for example 5 pmol? What is your opinion?

I designed these primers by Beacon Designer Software and they seem to work really well. (I performed gel electrophoresis, melt curve - everything is perfect). But why I still do not have good efficiency?

-Ewa-