# calculating total amount - in a pcr reaction (Jan/24/2011 )

can someone please check if this is right

given: 10ug of 200ng/ul DNA
find: how much total dna was added in a 25ul pcr reaction, if 5ul of dna was added into the reaction?

my calculations:

5ul*0.2ug/ul = 1ug, so 1ug of the total dna was added into the pcr reaction.

someone told me that total amount of dna doesn't change, but I am confused. I understand that dna on a mass level won't change and that is what the total amount of dna is showing, but if you add only 5ul of the dna into the pcr reaction, wouldn't you only be added 1ug of the total dna?

and is it better to know final concentration (ng/ul) rather than total amount in the reaction or does it depend on what you are after? when should one be used over the other?

-claritylight-

I do not understand your question or what you are saying really.

If you have 200 ng per µl and you take 5µl then you have 1000ng or 1µg ... in your tube.

I do not understand the 10ug of 200ng/ul DNA?

you mean that they started with 1µg in the PCR and because of you used the PCR you ended up with 10µg in the end?

And what about the mass wont change? the more DNA? the more mass???

You maybe mean that the volume wont change?

-pito-

given: 10ug of 200ng/ul DNA

so you had 50ul of 200ng/ul DNA to start? Making a total of 10ug?

You are correct in saying that 5ul of this solution would contain 1ug of DNA. But I don't really understand what you are asking. In saying that the total amount of DNA does not change, this is correct, but the concentration would change as you are adding it to a 25ul solution.

-philman-

Sorry if I have been so unclear. I'm just saying there is 10ug of DNA at concentration 200ng/ul, so yes there's 50ul volume. So 1ug or 40ng/ul of DNA is in the final pcr reaction. The person I was talking to had got me all confused so I wanted to make sure I was right first of all. They were saying something about the total amount of DNA not changing in the reaction.

My other question is why is this way wrong: (5ul/25ul)*10ug = 2ug. Since the ul cancel out, you are left with ug only. Is this used for anything?
I know it's correct if you did it using ng/ul: (5ul/25ul)*200ng/ul = 40ng/ul.

-claritylight-

claritylight on Fri Jan 28 02:45:02 2011 said:

Sorry if I have been so unclear. I'm just saying there is 10ug of DNA at concentration 200ng/ul, so yes there's 50ul volume. So 1ug or 40ng/ul of DNA is in the final pcr reaction. The person I was talking to had got me all confused so I wanted to make sure I was right first of all. They were saying something about the total amount of DNA not changing in the reaction.

My other question is why is this way wrong: (5ul/25ul)*10ug = 2ug. Since the ul cancel out, you are left with ug only. Is this used for anything?
I know it's correct if you did it using ng/ul: (5ul/25ul)*200ng/ul = 40ng/ul.

You first need to clear about terms used.
amount = ug or ng ... so on
volume = ul or ml ... so on
Concentration = ug/ul or ng/ul or ug/ml ... so on.

The term "mass" might be equal to amount coz mass refers to weight. But I think people don't use it often coz it's confusing.

The person you referred to; he is right. Total amount(ug or ng) of DNA in reaction doesn't change irrespective of PCR reaction volume. Only final concentration (ug/ul or ug/ul) changes.

If you add 5ul of 0.2ug/ul, there will be 1ug (1000ng) of DNA in your PCR reaction. That's what you called final amount.
If want to talk about final concentration, Yes, it does change.

If you PCR reaction volume is 20ul, the concentration will be 0.05ug/ul or 50ng/ul
If you PCR reaction volume is 50ul, the concentration will be 0.02ug/ul or 20ng/ul

Hope this helps.

P.S. Before you go on discussion, you guys should make it clear whether you all are on same page.
If you were talking about amount and others were talking about concentration, it'd be pointless arguing.

-N_L-