Dilution problem! - (Apr/28/2008 )

Hi all,

I have a basic dilution problem... I need to send some smaples for sequencing and i need to send in 100ng/ul of DNA in 10ul of sterile water. My stock DNA is 352ng/ul, now how do i dilute my stock to my desired ocncentration. please help me, pleaseeeeeee... i have tried to do it myself, but my sequencing results never gave any sequences so far, my PCR results were fine, but my sequencing results are just ****, i htink its becasue i have been seding too little or too much DNA. can someone please tell me how to dilute it please... Any help will be great appreciated.. thanks a lot...

C1V1=C2V2 where C1 is your stock concentration, C2 the desired concentration, and V2 the desired final volume, you can calculate how much of your stock solution you need=> V1.

in this case: 352*V1=100*10 V1=100*10/352=2.84ul so you need to add 2.84ul of your DNA to a final 10ul i.e 2.84ul DNA + 7.16ul of water (or whatever the buffer you use)

You may be the type of person that hates formulae.
If so then think about what you've got and what you want:

You want 10ul at 100ngul-1
You have got 352ngul-1


Compare what you've got with what you want:
352ngul-1 / 100ngul-1 = 3.52
That is, what you have is 3.52 times more concentrated than what you want.
So you have to dilute what you have 3.52 times.

You want 10ul, so you want to work out what number goes into 10ul 3.52 times (so that your DNA is diluted by a factor of 3.52). ie 10/3.52=2.84
So, you want 2.84ul DNA and dilute it with (10-2.84=) 7.16ul sddH2O or buffer or whatever.


If you break what you want and what you've got down into each part and then work there step by step then you'll do fine. This method is slower than relying on formulae but it might help you get sorted in your head what you need to do and what information you need to have to get to where you want to go.

A very simple method to calculate dilutions is the "mischungskreuz" (I dont know if there is an translation in english; should be something like dilution cross)

Its in my eyes the easiest way to calculate dilutions, as it is qick and easy :

concentration of solutuion 1 (352 ng/µL) - concentation needed (100 ng/µL) = the amount you need of solution 2 (252 parts of in this case water)

AND

concentartion needed (100ng/µL) - concentration of solution 2 (0 ng/µL) = the amount needed of solutuion 1 (100 parts of your 352ng/µL DNA solution)


And if you take 10 µL as final concentration you get: (10 µL / 352 parts) * 100 = 2,84 µL of solution 1
and (10 µL / 352 parts) * 252 = 7,15 µL of water


online version with explanation

hope you can understand my explaination as it is difficult without drawing it .... for a graphic of this method look at the german wikipedia searching "mischungskreuz" there is a nice picture of this method!