Creating qPCR dilutions

5 posts / 0 new
Last post
potato_head
potato_head's picture
Creating qPCR dilutions

Hi everyone,

I need some help setting up a qPCR experiment.
I need to make up dilutions for a qPCR reaction standard curve using the following instructions:

You are provided with a tube that contains 500,000 molecules per microlitre (uL). Set up 4 reaction tubes containing 10^6, 10^5, 10^4 and 10^3 molecules respectively (i.e. 10 fold dilutions). Note that accurate pipetting below 0.5uL is not possible.

Could someone please expain how I can achieve this?

Ivan Delgado
Ivan Delgado's picture
Hi potato_head,

Hi potato_head,

I think you may have a problem here. The notation "10^6" implies a 1 followed by 6 zeros, so it should mean 1,000,000 molecules per microliter. Since you only have a tube containing 500,000 molecules per microliter you cannot really dilute that into 1,000,000 molecules per microliter, since this last solution contains more molecules per microliter than the solution you have to begin with. I guess you could concentrate your 500,000 molecules per microliter solution and get a 1,000,000 molecules per microliter solution, but I am guessing that is not what you are being asked for. 

As for a 10^5 solution, that is what you have to begin with. To obtain the rest you simply make 1:10 dilutions of each. 

Cheers

anna F
anna F's picture
Hi everyone :)

Hi everyone :)

I need some help with a nanodrop and dilution DNA sample. after a measure in 1ul I have a 1700ng/ul.
It does mean that all volume of my samples ( 100ul ) is in a concentration - 1700ng/ul? I need to dilute my sample to 15ng/ul.  Could some please explain me?

Rajeshwari patel
Rajeshwari patel's picture
You have concentration 1700ng

You have concentration 1700ng/uL  and you have total 100ul i.e you have total 170000ng DNA i.e 170 ug of DNA

For getting concentration 15ng/uL  you have to do serial dilution like

first 5 fold dilution       2 uL( DNA)+ 8 uL( buffer)  which results in 340ng/uL
Than 10 fild dilution   5uL( DNA)+ +45 uL( buffer)   which results in 34 ng/uL
than 2.3 fold dilution 22uL( DNA)+28uL( buffer)  which results in 15 ng/uL.

I think This will hepl you

Regards
Rajeshwari

potato_head
potato_head's picture
Thanks for your help Ivan, I

Thanks for your help Ivan, I think I've got it sorted now.