Primer dilution

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AmB
AmB's picture
Primer dilution

 How do I make 20micromolar primer stock from the lyophilized primers?Eg. If the label says 46.4nmols?

Ivan Delgado
Ivan Delgado's picture
Hi AmB,

Hi AmB,

Unfortunately your calculations are not correct. If you resuspend 32.2 nmoles of dry primer in 64.4 microliters of water you will get a 500 micromolar stock.

Here is the procedure I typically follow when resuspending and diluting primers: 

Step 1: resuspend your X nanomoles of dry primer with 10X microliters of water/buffer to get a 100 micromolar stock of primer. In your case resuspend your 32.2 nmoles of dry primer with 322 microliters of water. It is a good idea to prepare a 100 micromolar stock first because primers at this higher concentration are more stable.

Step 2: dilute your 100 micromolar stock 1:10 to get a 10 micromolar stock. Since you are looking for a 20 micromolar stock, instead of performing a 1:10 diltuion you would perform a 1:5 dilution. For example you could take 10 microliters of your 100 micromolar stock and mix it with 40 microliters of water to get a 20 micromolar stock.

To your original question, if you condense the math described above, instead of 64.4 microliters you would need to add 1,610 microliters of water to your 32.2 nanomoles of dry primer to get a 20 micromolar stock. If that is what you need to do, and if you rather not follow the 2-step procedure described above, that is the one-step way to get a 20 micromolar stock from 32.2 nanomoles of dry primer. 

Cheers

AmB
AmB's picture
 Hey Ivan,

 Hey Ivan,
Thanks for the quick reply.....yes, I get my maths error....just worked that out myself too..:-)...and I think I posted two things together....anyways.....thanks for helping clear my doubt.....

Best,
AmB

Ivan Delgado
Ivan Delgado's picture
Anytime. 

Anytime. 

Good luck getting your PCRs working!