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Dilution of Stock DNA

genetics

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4 replies to this topic

#1 jlr138

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Posted 30 August 2012 - 08:53 PM

Hello I am about to run a PCR and am trying to dilute the DNA samples but the volume is coming up too short

I have stock dna of about 10 ng/ul and need to prepare a 6 ul of dna at 3ng/ul

first i used c1v1=c2v2 and the result came as 1.8 ng/ul of dna this is too small so I need to run a serial dilution, and at this point I am stuck. I tried first using

D= Cf/Ci
= 3/10 and then am stuck at this point I dont understand how I can make a 3/10 serial dilution, all that I have seen online is 1/X example 1/30 which makes sense I can do 1 ng/ml of dna + 9 ml of sterile water this equals to 1/10 and then take 2 ul of dilution 1 and add to 4 ul of sterile water to get to my target volume of 6 ul but starting with 3/10 is very confusing? Help please!

#2 mdfenko

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Posted 30 August 2012 - 08:58 PM

your error was in not canceling out units. your result was actually to use 1.8ul of 10ng/ul to make 6ul at 3ng/ul (which is correct).
talent does what it can
genius does what it must
i do what i get paid to do

#3 jlr138

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Posted 30 August 2012 - 09:13 PM

thanks,

can I ask another ? i have stock dna @ 20 ng/ul need same amount as above, and the answer I get is .9 ul and this is too small for the micro pipettes how would i perform a serial dilution if I start with 3/20?

#4 pito

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Posted 31 August 2012 - 05:00 AM

you need to go from 20ng/µl to 3ng/µl, right?

And what endvolume do you need?

An example:

take 1.8µl from the stock, add it to 4.2µl dilutant => makes 36ng for 6µl or 6ng per 1µl.

if you then take 1µl of this and add this in 1µl dilutant, you have 6ng in 2µl or 3ng per µl


Or simple math:

20 ng/µl ==> 3ng/µl , means x(3/20) , meaning you take 3µl from stock and add this to 17µl dilutant (totaling 20µl). Means: 3µl with 20ng/µl, is 60ng in total in endvolume of 20µl , means 60ng/20µl = 3ng/µl


PS. it seems you did not understand mdfenko, because if you did, you would know that if you need 1.8µl from 10ng/µl and add this to 4.2 to make 3ng/µl, you would also now that you simple could to the same thing followed by diluting it in half (1/2) since your stock now is twice (20ng compared to 10ng).
Its the same as taking 0.9 , only 0.9 is too small to take from the stock.. so you take dilute it after a first dilution rather then diluting it at once.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#5 mdfenko

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Posted 31 August 2012 - 09:26 PM

thanks,

can I ask another ? i have stock dna @ 20 ng/ul need same amount as above, and the answer I get is .9 ul and this is too small for the micro pipettes how would i perform a serial dilution if I start with 3/20?

you can pipet 1.8ul but not 0.9?

okay, then dilute the 20ng/ul 1:1 (to 10ng/ul) and take 1.8ul.
talent does what it can
genius does what it must
i do what i get paid to do





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