I have 100 uM stock of forward+reverse primer mix. I have made a 10uM working concentration for the use in PCR. How much volume of this mix should i add to a 50 ul reaction volume so that i have a final concentration of 0.2uM of forward as well as reverse primer?
Hi there,
If you don't set the volume of PCR reaction mix, one can't answer the question in term of volume! Dilution factor will be 50 starting from your 10µM solution.
Hi. First of all, I quite not understand why you have the two primers in the same mix, I always order them separately. Second, if you have 10 uM working tube then you have 5 uM of each primer (in theory), and you should add, for a final volume of 50 ul, 1 ul of each primer. Thus, add 2 ul of the primers mix for a final PCR volume of 50 ul. Good luck! :)
Hi Dominique , the total volume of my master mix is 50ul.
Hi Izaskun, actually i have not ordered the primers yet. But for the initial standardization i got the mix from a colleague. And while setting the reaction , it was pretty confusing
I agree with both the answers given above. First you should define reaction volume then you can take the required amount of primers from stock using the formula C1V1=C2V2.
Hi Bhoomika,
You should have said: the total 'reaction volume' in each PCR tube is 50 uL, not the total 'master mix'. Master mix usually means that some PCR ingredients have been pre-mixed and can dispense into your PCR tubes, then you add different DNA template samples. See attached picture for 'Master mix'.
use a basic formula c1V1=c2V2 where c1 is initial stock concentration, c2 - target concentration (10uM fo example), V2 - target volume (for working solution), V1 - should be found, i.e. V1=(c2V2)/c1. Formula works well when V1
Bhoomika wrote: I have 100 uM stock of forward+reverse primer mix. I have made a 10uM working concentration for the use in PCR. How much volume of this mix should i add to a 50 ul reaction volume so that i have a final concentration of 0.2uM of forward as well as reverse primer?How should i calculate the amount of primers to be added from a primer mix?
the PCR volume is correctly indicated, from what you say, the primers should be 10 uM each in your dilution. As some people told you before, you should use the formula C1V1=C2V2, where:
10uM XuL=0.2uM 50uL x=1 uL
@ M N Manoj,
Should be 0.4 uL from the 50 uM stock (0.2 uL for the F and 0.2 uL for the R) for 50 uL final reaction volume, I think.
@ Paola,
X=1 uL, that is for one primer. In a PCR reaction, 2 primers are needed, so you need 2 uL from the 10 uM stock.
Hi Joana. If we take 1ul from the primer mix , then we will have 0.5ul of forward and 0.5ul reverse primer . According to this formula, the concentration required for each primer will be reduced to half.Though i am not sure but I think 2ul of the primer mix should be added. Please let me know if i am wrong somewhere conceptually.
Hi, Bhoomika.
When I have several targets to be studied I also prepare a working solution of the two primers in a mix.. to do this I start from the two 100 uM stocks and prepare the dilution by putting, in proportion, 1 ul of the fw primer, 1 ul of the rv, 8 ul of nuclease free water. In this way, each of the primers will be diluted 1:10 (=10 uM) and we can say the primers are 10 uM each. In your case, it depends on how the mixed stock solution was prepared :
1) you resuspended
My message was cut in half!
Anyway, Joana is correct : if you mix 1 volume 100uM of the fw with one volume 100uM of the rv, actually you get 50 uM of each primer, because you dilute each primer with the volume coming from the othe one:
let's say you use 10 ul of A and 10 ul of B. 10 of A is now in 20 ul, so its concentration is halved, and the same for B... I hope it is now clear...
Most PCR reactions use 0.1−0.5 μM primer. Assuming a maximum concentration of 0.5 μM and a reaction volume of 20 μL, each reaction will require 10 pmol oligonucleotide primer. Bhoomika Sharma
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