Lidstrom:Diluting Primers: Difference between revisions

Revision as of 19:10, 18 January 2012

Primer Dilutions: People generally dilute dehydrated primers that arrive in the mail to 100uM, and use a working concentration of 10 uM.

To get 100uM: dissolve dehydrated primer with in 10 uL per nmole:
- 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

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

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 Primer Dilutions:
 People generally dilute dehydrated primers that arrive in the mail to 100uM, and use a working concentration of 10 uM.
+== Prepare 100 uM stock ==
+*To get 100uM: dissolve dehydrated primer with in 10 uL per nmole:
+**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
-To get 100uM: dissolve dehydrated primer with in 10 uL per nmole:
+==Prepare primer for use==
-For eg., for 25 nmoles of primer add 250 ul of water
+*Dilute this stock 1:10, to give a concentration of 10 uM
-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
-To prepare primer for use:
+==Use in PCR==
-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.
-From this, use __ ml in a typical __ uL PCR reaction. This will give you a final concentration of __ pmoles in a PCR reaction.