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Primer concentration


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#1 muc77

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Posted 21 July 2011 - 02:51 AM

Hi,


I惴 a little confused caused by Primer concentration dilution.
Our stock concentration is 20然, working con. Is 2然.
How do I dilute this:
Primer from vendor: 100然 (dilutet by me)
Now I take 20無 PrimerF and 20無 PrimerR and fill up to 200無 with water. That愀 our 20然 stock.
To get the working concentration I take 1無 stock + 9無 water-> 2然.

But I can dilute 2無 PrimerF and 2無 PrimerR and fill up to 200無 with water. That愀 our 2然 stock.
So I can get directly my 2然 concentration.

Now I need 500nM working concentration and I use 2無 PrimerF and 2無 PrimerR and fill up to 640無.

Make that sense?


Greetings,
Mathias

#2 Trof

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Posted 21 July 2011 - 08:26 AM

To be sure we understand each other, there are some definitions:
1 然 equals 1 pmol/痞
Stock concentration is what you keep safe for diluting to working concentration, better rather concentrated because that prevents degradation. 100 然 or 200 然 is usually used as a stock concentration.
We use 100 然.

Working concentration is what you pipet from to PCR, in volume corresponding to the frequency of usage (for 10 reactions every week make less than for 10 reaction every day) and concentration corresponding to the desired final concentration in PCR reaction (ideally pipet around 1 - 2 痞 of working concentration to a single reaction, if you do really many samples at once, the volume can be smaller and concentration higher).
We use 10 然 each to use 1 痞 of it for single reaction.

Final concentration in PCR reaction is what you get when you pipet your working concentration to the desired PCR volume. That is usualy recomended to be 0.3 - 1 然, with 0.5 然 (500 nM) of each as a starting concentration.
As we pipet 1 痞 of our 10 然 each (10 pmol/痞) primers to 20 痞 reaction, final concentration is 10/20 = 0.5 uM of each.

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Now your stock concentration (20 然) is pretty low, I would suggest to use higher. Working concentration seems also low for classic PCR, but you may have reason for it. But as you say you need 0.5 然 working concentration I suspect you maybe confuse working concentration and final concentration in PCR reaction. So what concentration (final) do you need in your PCR?

Your calculations of stock concentrations are wrong. If you have 20 痞 of 100然 primer in 200 痞, it's 10然, not 20然. You need to calculate 20 然 for each, not for both together (or use the primers separately, not mixed together). Correct is to take 40 痞 of each primer and fill up to 200. The calculation of 2然 and 500nM concentration has the same mistake, so it's also wrong. And I wouldn't recommend to have 640 痞 of working concentration unless you use up this amount say in one month. It's better to have smaller volumes of working concentration so you don't store it too long and for changing to freshly made working concentration in case you suspect contamination.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#3 Bea Kerr

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Posted 21 July 2011 - 04:29 PM

I'm confused as to what your final concentration needs to be also.

Use C1V1=C2V2. If you're still confused try using the "Dilute a stock solution" calculator on http://www.graphpad....olarityform.cfm
It will give you the answer, but then also try and work it out for yourself so it makes sense.

#4 muc77

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Posted 21 July 2011 - 09:03 PM

Hi,


ok, you are right. My mistake was that I calculate 20然 for both primer together not each.

To be sure we understand each other, there are some definitions:
1 然 equals 1 pmol/痞


Yes indeed.

I try again:

Stock: 100然
Working: 10然


I think I get it.

For further dilutions I take 20無 100然 in 200無 to get 10然, right? And 40無 100然 in 200無 for 20然.
And for both primer in a dilution to get a 10然 working concentration: 20痞 100然 F-Primer and 20痞 100然 R-Primer and fill up with 160痞 water to 200痞 total vol. Right?
And for both primer in a dilution to get a 20然 working concentration: 40痞 100然 F-Primer and 40痞 100然 R-Primer and fill up with 120痞 water to 200痞 total vol.

The more I think about the more confusing is it.

Thanks a lot!!!

#5 Trof

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Posted 22 July 2011 - 12:43 AM

For further dilutions I take 20無 100然 in 200無 to get 10然, right? And 40無 100然 in 200無 for 20然.
And for both primer in a dilution to get a 10然 working concentration: 20痞 100然 F-Primer and 20痞 100然 R-Primer and fill up with 160痞 water to 200痞 total vol. Right?
And for both primer in a dilution to get a 20然 working concentration: 40痞 100然 F-Primer and 40痞 100然 R-Primer and fill up with 120痞 water to 200痞 total vol.

Yes, that's right.

If it helps you, for this type of calculations I usually imagine the concentration as number of moles floating in corresponding volume (I do actually see them floating in my mind, but that's not required ;) ). Since you mostly work in 痞 volumes, I "convert" 10 然 to 10 pmol/痞 and imagine 10 pmoles floating in one 痞. So if you take 20 痞 of this stock, you just calculate 10*20=200 that's the number of pmoles floating in 20 痞 I took out. Then I look at the final volume of dilution. I have the final volume say 200 痞, so there will be 200 pmoles I took earlier floating in 200 痞 and to get back the concentration I divide it by the number of 痞s, 200/200=1 that is 1 pmole in 痞 which equals 1 然.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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