I want to calculate my primer concentration to be able use the Tm calculator on New England Bio lab. I have primers and I need to amplify by using Phusion High-Fidelity DNA polymerase but I do not know how to calculate my primer concentration (nM).
https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator
Hi Ayat,
You must know that oligo synthesis companies always give you a synthesis report sheet where you can find several features for you oligo (i.e. sequence, yield, mw, tm-theoretical, etc). The nmol yield can be used to calculate concentration for your oligo. To get a standard 100uM concentration, you must add the nmol*10 volumen (uL). For instance, if your oligo was synthesized and the nmol yield is 44.2, then you must add 442uL of nuclease-free water to get 100 uM concentration. From this on, you can easily to calculate work solutions (i.e 10 uL) by diluting 1/10 the stock, and so on!
Good luck!
Hi Ayat,
When you received your primers (eurofins, sigma, mwg) they sent you a paper when it is writing who many µL of water you have to put on each tube. Normaly at the end you have 100pmol and I used my primers at 10pmol for the PCR with the Phusion.
I used to make 1/10 dilution of my primers before using them.
Hi Ayat,
You must know that oligo synthesis companies always give you a synthesis report sheet where you can find several features for you oligo (i.e. sequence, yield, mw, tm-theoretical, etc). The nmol yield can be used to calculate concentration for your oligo. To get a standard 100uM concentration, you must add the nmol*10 volumen (uL). For instance, if your oligo was synthesized and the nmol yield is 44.2, then you must add 442uL of nuclease-free water to get 100 uM concentration. From this on, you can easily to calculate work solutions (i.e 10 uL) by diluting 1/10 the stock, and so on!
Good luck!
Once you have made the stock concentration use N1V1 = N2V2 formula.
If the synthesis report is lost, you can still see the amount of nmoles on the small tube where the lyophlized primer is. If you are older, ask a younger lab member to read it for you, cause it is usually written in tiny letters... Another option is to send an email to the provider of the primers and ask to send you a new report.
I have uploaded a very simple spreadsheet for the calculation of the primers concentration in the final PCR reaction volume. Method A spreadsheet that calculates primer concentration in the fi...
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