Hello,
I am using a gene speciic primer to perform reverse transcription. I need to use 20pmols of my primer in a final reaction volume of 20ul. Is 20pmol interpreted as 20pmol/uL (20uM) in RT, or am I to find the molarity of 20pmols in 20ul? If I take the later approach, my final concentration is 1uM, which seems too small of a concentration to add to my solution. I'm struggling with interpreting the use of moles in most papers when it comes to primer concentrations and knowing exactly what final concentration they used.
Hello Camille,
nmol or pmol are amounts (quantity) not concentration!
20 pmol need to be added to your final 20µl reaction volume.
Here is a trick,
1. Find how much amount of primer do you have (On specification sheet or primer vial label), it is mentioned in 'nmol'.
2. Covert it into pmol.
e.g. my oligo is 28.7nmoles = 28700pmol (for 100µM add 287µl ddH20)
3. All you need to find is how much volume you need to take from 287µl (having 28700pmol) so that you could add to your (20 - primer) µl rxn volume.
e.g. workout this simple equation;
1pmol is contained in 287/28700 µl
20pmol is contained in (287/28700)*20 =0.2µl (100µM)
P.S. Please note that 0.2µl that's going to be added is still 100µM.
We generally use 10µM for RT. You can calculate accordingly.
Hope it helps.
Regards,
JP
Hello Camille,
nmol or pmol are amounts (quantity) not concentration!
20 pmol need to be added to your final 20µl reaction volume.
Here is a trick,
1. Find how much amount of primer do you have (On specification sheet or primer vial label), it is mentioned in 'nmol'.
2. Covert it into pmol.
e.g. my oligo is 28.7nmoles = 28700pmol (for 100µM add 287µl ddH20)
3. All you need to find is how much volume you need to take from 287µl (having 28700pmol) so that you could add to your (20 - primer) µl rxn volume.
e.g. workout this simple equation;
1pmol is contained in 287/28700 µl
20pmol is contained in (287/28700)*20 =0.2µl (100µM)
P.S. Please note that 0.2µl that's going to be added is still 100µM.
We generally use 10µM for RT. You can calculate accordingly.
Hope it helps.
Regards,
JP
- Agreed
- the standard amount of primer you typically add to PCR of cDNA, i.e. RT PCR (Not qPCR) is 1ul of primers at 10uM or 20uM
- At this concentration this equated to either 10 or 20pmoles of each primer (therefore say 10uM = 10pMoles/ul)
- For very high copy number genes you can get away with 1ul @ 5uM; that is 5 pmoles total amount (= 5pmoles/ul concentration = 5uM) but not less than that; 10-20uM is a good starting point for new primers and/or gene with an unknown copy number in your extract
J.P. Mandal and Laurence Stuart Dawkins-Hall thank you! This really helps.
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