Lidstrom:Diluting Primers

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(New page: Back to: Lidstrom Protocols Primer Dilutions: People generally dilute dehydrated primers that arrive in the mail to 100uM, and use a working concentration of 10 uM....)
Current revision (11:12, 25 May 2012) (view source)
(Prepare 100 uM stock)
 
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Primer Dilutions:
Primer Dilutions:
People generally dilute dehydrated primers that arrive in the mail to 100uM, and use a working concentration of 10 uM.  
People generally dilute dehydrated primers that arrive in the mail to 100uM, and use a working concentration of 10 uM.  
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Store diluted primers at -20oC.  Lyophilized (water-free) primers are quite stable at room temperature.
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== Prepare 100 uM stock ==
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*To get 100uM: for ever nmole, add 10 uL:
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**Example: for 25 nmoles of primer add 250 ul of water
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***This will give you a concentration of 0.0001 M or 0.1 mM
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****(1 nmole/10uL) = (1 nmole/10uL)*(10^6 uL/L)*(10^-9 mole/nmole) = 0.0001M = 0.1 mM = 100 uM
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*The most common working concentration is 10 uM (for PCR) or 5 uM (sequencing with GeneWiz)
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==Prepare primer for use==
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*Dilute this stock 1:10, to give a concentration of 10 uM
   
   
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To get 100uM: dissolve dehydrated primer with in 10 uL per nmole:
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==Use in PCR==
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For eg., for 25 nmoles of primer add 250 ul of water
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*From this, use __ ul in a typical __ uL PCR reaction. This will give you a final concentration of __ pmoles in a PCR reaction.
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This will give you a concentration of 0.0001 M or 0.1 mM
+
-
(1 nmole/10uL) = (1 nmole/10uL)*(10^6 uL/L)*(10^-9 mole/nmole) = 0.0001M = 0.1 mM = 100 uM
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-
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To prepare primer for use:
+
-
Dilute this stock 1:10, to give a concentration of 10 uM
+
-
+
-
From this, use __ ml in a typical __ uL PCR reaction. This will give you a final concentration of __ pmoles in a PCR reaction.
+

Current revision

Back to: Lidstrom Protocols

Primer Dilutions: People generally dilute dehydrated primers that arrive in the mail to 100uM, and use a working concentration of 10 uM. Store diluted primers at -20oC. Lyophilized (water-free) primers are quite stable at room temperature.

Prepare 100 uM stock

  • To get 100uM: for ever nmole, add 10 uL:
    • Example: for 25 nmoles of primer add 250 ul of water
      • This will give you a concentration of 0.0001 M or 0.1 mM
        • (1 nmole/10uL) = (1 nmole/10uL)*(10^6 uL/L)*(10^-9 mole/nmole) = 0.0001M = 0.1 mM = 100 uM
  • The most common working concentration is 10 uM (for PCR) or 5 uM (sequencing with GeneWiz)

Prepare primer for use

  • Dilute this stock 1:10, to give a concentration of 10 uM

Use in PCR

  • From this, use __ ul in a typical __ uL PCR reaction. This will give you a final concentration of __ pmoles in a PCR reaction.
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