final concentration - (Feb/11/2011 )

If there are 3 tubes of PCR reaction at concentration each of 20ng/ul, and I pool them together, will final concentration be 6.6ng/ul (20/3=6.6) due to DNA concentration being diluted down?

claritylight on Fri Feb 11 21:55:20 2011 said:

The concentration will be the same.

Ex: if you have 5 stones in each 1 liter bottle and you have 3 bottles, you put those bottles in one big 3 liter bottle: how many stones do you have? 15 , and how many liters? 3 , wich makes 15 stones in 3 liter or 5 stones in 1 liter..
(each stone has the same mass, I just picked stones, its easier to get the idea, imagine the stones being your DNA).

A concentration is only diluted if you add more solvent (liquid here, water for ex.).

And since the three tubes have the same concentration it doenst matter if you add them all in 1 big tube, it stays the same.
(even if the volume is different, ex: 1 bottle with 2 liters, meaning 10 stones since the concentration is 5 stones for each liter... 2 other bottles are Again 1 liter with 5 stones, put them all in one bottle: 4 liters with 20 stones... )


And with the DNA its just the same: 20ng for each µl, so if you add those 3 tubes all in one tube: each µl will still be 20ng...
(ex, if each tube is 10µl, you will have: 10 µl + 10 µl + 10µl = 30 µl and in that 30 µl you will have 20 ng x 30 (since you have 30µl and its 20ng for each µl) = 600 ng or 600 ng/30 µl = 20 ng/µl).

Thank you pito. Will this be same or different for primers, if I have forward and reverse primer at 20uM each, and I mix them together, they will be at 20uM final as well?

claritylight on Sat Feb 12 14:18:49 2011 said:

What do you think?


1 M means: 1 mol per liter...
Think like with the stones..
1 stones in each liter... I have have 2 bottle like that and I put them into 1 bigger bottle, how many stones (moles) will I have then?

1 liter of 20µM + 1 liter of 20µM, how many µM will that be in the 2 liter bottle? And if you recalculate it to 1 liter after putting them together?


Always remember: concentrations dont change as long as you dont dilute it.... So if you add X times the same concentration together, the concentration stays the same...

Look it like this: if you had a tube containing 20µl with a 10ng/µl solution, how much ng would you have if you took 5µl or 15 µl ? And how much would this be in concentration...?

It goes the other way around to: adding 5 µl of a 6ng/µl concentration together with 8µl of a 6ng/µl concentration is in the end still a 6ng/µl concentration (do the math... check it).



It doenst matter wether we are talking about primers, stones, sugar, DNA, or whatever it is you have there .....

if you mix 1ml of 20uM forward primer with 1ml of 20uM reverse primer then you will have 2ml of 20uM primer mix but 10uM each primer.

mdfenko on Mon Feb 14 21:40:16 2011 said:

Damn, didnt concider the reverse and forward part.

My post is off course not right if you look at the primers each individually.

You will have diluted your forward and reverse primers and thus making it a 10µM of each or 20µM "primers" in general.