Difference between revisions of "Lidstrom:Diluting Primers"

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(Prepare 100 uM stock)
 
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== Prepare 100 uM stock ==  
 
== Prepare 100 uM stock ==  
*To get 100uM: dissolve dehydrated primer with in 10 uL per nmole:
+
*To get 100uM: for ever nmole, add 10 uL:
 
**Example: for 25 nmoles of primer add 250 ul of water
 
**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
 
***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
 
****(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==
 
==Prepare primer for use==
 
*Dilute this stock 1:10, to give a concentration of 10 uM
 
*Dilute this stock 1:10, to give a concentration of 10 uM

Latest revision as of 09:12, 25 May 2012

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.