Lidstrom:Diluting Primers: Difference between revisions
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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. | ||
Store diluted primers at -20oC. Lyophilized (water-free) primers are quite stable at room temperature. | |||
== Prepare 100 uM stock == | == Prepare 100 uM stock == | ||
*To get 100uM: | *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 | ||
==Use in PCR== | ==Use in PCR== | ||
*From this, use __ | *From this, use __ ul in a typical __ uL PCR reaction. This will give you a final concentration of __ pmoles in a PCR reaction. | ||
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
- This will give you a concentration of 0.0001 M or 0.1 mM
- Example: for 25 nmoles of primer add 250 ul of water
- 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.
