# Lidstrom:Diluting Primers

(Difference between revisions)
 Revision as of 22:09, 18 January 2012 (view source) (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....)← Previous diff Current revision (12:12, 25 May 2012) (view source) (→Prepare 100 uM stock) (3 intermediate revisions not shown.) Line 3: Line 3: 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. + 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 - To get 100uM: dissolve dehydrated primer with in 10 uL per nmole: + ==Use in PCR== - For eg., for 25 nmoles of primer add 250 ul of water + *From this, use __ ul in a typical __ uL PCR reaction. This will give you a final concentration of __ pmoles in a PCR reaction. - 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: + - 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.