PCR basics - I'm new to all things lab and need some help! (Apr/21/2011 )

Hi all,

I've recently started a PhD and my background is not in science as such, so I'm finding it difficult to grasp basic concepts - so any help would be much appreciated (I'm sorry if these questions are incredibly simple).

Concentrations really confuse me as I've never learned about moles etc. What I'm trying to work out at the moment is how to calculate how much stuff I've put into a PCR. For example, in a 25 microlitre qPCR reaction I have included 0.25µl of probe (working concentration 5µM)- so how many µM of probe are in there?! I get even more confused when different units are involved - such as 2.4µl MgCl2 (stock concentration 25mM)in a 20 µl reaction. I know these questions are probably very stupid to a person with half a science brain cell, but I'm getting myself so confused - so if anyone could explain the principle of this in very basic terms I would appreciate it a huge amount!

Thanks every so much! :-)

-katcvet-

katcvet on Thu Apr 21 16:55:45 2011 said:

if you have 0.25µl of a 5µM probe in 25µl you have done a 1:100 dilution. that means that there is also just 1/100 of 5µM in your reaction. 5µM = 5000nM and 5000/100 = 50.
so, your PCR reaction has a 50nM concentration of probe. it is that easy.

With this M/moles it is important not to confuse a concentration (like 5µM) with the quantity of a substance like 5µmol.

5µM means that in 1L of your solution are 5µmol of substance.

5µmol means if you put this amount of substance in 1L of water, the solution is 5µM.

I hope this helped.

-tea-test-

Concentrations really confuse me as I've never learned about moles etc. What I'm trying to work out at the moment is how to calculate how much stuff I've put into a PCR. For example, in a 25 microlitre qPCR reaction I have included 0.25µl of probe (working concentration 5µM)- so how many µM of probe are in there?! I get even

tea-test is right, but I would like the adress you to the math behind it, especially because you dont seem to know the basics enough.

0,25µl = 0,25 *10^-6 liter
5µM = 5 * 10^-6 Moles/ liter (M= moles/liter)

Thus: 0,25 *10^-6 liter * 5 * 10^-6 Moles/ liter = 1,25 * 10^-12 moles

You have this in a tube with an end volume of 25 µl wich is 25 * 10^-6 liter, thus you have 1,25 * 10^-12 moles / 25 * 10^-6 liter

wich in turn is: 0,05 * 10^-6 moles/liter or 5 * 10^-8 moles/liter or 50 * 10^-9 moles/liter and this is 50nM

So its indeed very easy to use what tea test did: its a 1/100 dilution.. and you can use this, but always keep the calculation I did in your mind whenever you have doubts about what kind of dilution it is.

In general:

At first you had 0,25µl, the volume you took, this contains 1,25 * 10^-12 moles and then you add extra volume untill you reach 25µl , so the number of moles stay the same... you simply dilute it untill you have 25µl in total
So you dilute the amount of moles with 1OO, 5µM becaumes 50nM (5µMoles/liter, diluted 100 times is 5µMoles/100liter or 5µ * 10^-2 Moles /liter ==> 50nmoles/liter.

This is how tea test did the math, its fast and easy....
But if in doubt: do the math, the entire calculation. It will be longer but it can help and in the end you are so familiar with it that you dont need to think about it.

BTW: you are doing a PhD that involves PCR but did have basic maths? What bachelor/master did you do then? And is it a PhD in the field of molecular biolgy or something like that?

-pito-

Thank you both so much for your replies, that's really helped me

I'm doing a PhD in virology/molecular biology. I'm actually a veterinary surgeon (not a 'true scientist'!); I did a BSc in vet pathology too which involved a three month lab project - but that was a long long time ago! So whilst I can tell you what dose of antibiotic you need for your cat, I am completely hopeless when calculations get any more complicated than that!

-katcvet-