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Primer Dilution Confusion

Primer Dilution Confusion - PCR - Polymerase Chain Reaction Forum

Primer Dilution Confusion - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 08-13-2012, 11:45 PM
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Default Primer Dilution Confusion



I just read over our labs primer dilution protocol. I am trying to figure out what our primer concentration is in nM. I would like to know if someone could check my math.

My primers came dried at a concentration of 23.5nMoles.
-----------------------------------------------------------------------------------------
(23.5nmoles/1175ul water)=.02nmoles/1 ul
(.02nmoles/1 ul)*(1000)=20pmoles/ul

*20pmoles/ul is our stock solution.*

((44ul)*(20pmoles/ul))/500 ul water = 1.76 pmoles/ul

*1.76 pmoles/ul is our working solution.*

1.75 pmol in 1μl = 1750 pmol in 1 ml = 1,750,000 pmol per litre = 1750 nmol/l
Since mol/l is the same as M: Then 1750 nmol/l = 1750 nM.
-----------------------------------------------------------------------------------------

From what I have read normal concentration for realtime pcr is 50-300nMolar. If this is true and my calculations are correct then I need to significantly decrease the concentration of our primers that our current protocol calls for.
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Old 08-17-2012, 05:53 PM
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Default Re: Primer Dilution Confusion

I would just like someone to check my math. If the math is correct then is this concentration too high?
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Old 08-19-2012, 03:53 PM
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Default Re: Primer Dilution Confusion

Quote:
Originally Posted by ah535 View Post
I just read over our labs primer dilution protocol. I am trying to figure out what our primer concentration is in nM. I would like to know if someone could check my math.

My primers came dried at a concentration of 23.5nMoles.
-----------------------------------------------------------------------------------------
(23.5nmoles/1175ul water)=.02nmoles/1 ul
(.02nmoles/1 ul)*(1000)=20pmoles/ul

*20pmoles/ul is our stock solution.*

((44ul)*(20pmoles/ul))/500 ul water = 1.76 pmoles/ul

*1.76 pmoles/ul is our working solution.*

1.75 pmol in 1μl = 1750 pmol in 1 ml = 1,750,000 pmol per litre = 1750 nmol/l
Since mol/l is the same as M: Then 1750 nmol/l = 1750 nM.
-----------------------------------------------------------------------------------------

From what I have read normal concentration for realtime pcr is 50-300nMolar. If this is true and my calculations are correct then I need to significantly decrease the concentration of our primers that our current protocol calls for.
Looking closer at your maths.
(23.5nmoles/1175ul water)=.02nmoles/1 ul
(.02nmoles/1 ul)*(1000)=20pmoles/ul


I thought 1 nmol = 1000 umol. How did you end up with pmol?? Therefore 0.02nmo/ul should be 20 umol/ul.

I will say this: Most primers usually come with a primer sheet that tells you how much to water to add to get u100 M. That's the most common concentration for stock solution. For regular primers around 18-25 bp, the amount of water that is suggested rarely exceed 1000 ul. Please don't use your own calculations using the primer formula weight/dry weight etc.

From the stock concentration, then we will dilute the 100 M stock 10x to get 10uM of working concentration.

I will strongly advice against preparing weird concentrations for stock and working, unless you really know what you're doing. I find it very unlikely that you will necessarily need to use 1.76 M in a PCR.
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  #4  
Old 08-20-2012, 05:19 AM
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Default Re: Primer Dilution Confusion

Thank you for the reply. I thought something was off. I would like to start diluting our primers like every other lab does.
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Old 09-03-2012, 06:42 AM
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Default Re: Primer Dilution Confusion

Your math is correct. Regarding realtime PCR if you need 50nM concentration of your primer this means 0.05 pmol/uL of your total volume PCR reaction. So for 25 uL PCR reaction you will need to add 1.25 pmols of your primer. So if you diluted your stock of 20 pmol/ul to be 1 pmol/uL you will need 1.25 uL of each primer in your PCR reaction
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