DNA Solution - Some Queries

Duncan Clark via methods%40net.bio.net (by blackhole from abuse.plus.com)
Tue May 13 07:29:49 EST 2008


Historians believe that in newspost 
<7e3e00a7-f5c2-4636-bad7-98263e35f13a from t54g2000hsg.googlegroups.com> on 
Tue, 13 May 2008, lsl <sioklian from gmail.com> penned the following literary 
masterpiece:
>1) We purchase the synthetic oligonucleotides and they claim to have
>100uM of DNA. Is this number reliable for each and every batch?

Yield varies batch to batch, oligo to oligo.

>2) If what is claimed by the oligo supplier is not really correct,
>usually what is the method you used to prepare certain concentration
>of DNA solution (say 10uM)?

Go to an oligo calculator site say IDT or any primer design program.

http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/

Feed in your oligo sequence and it will come back with a figure of 
nmol/OD and ug/OD i.e. 1 OD260 of a 20mer I juts checked here is 
calculated as 4.82nmol or 30.2ug.

>3) How to use UV-vis to check the concentration of a DNA solution and
>how to calculate the amount of DNA present in the solution? If
>possible, any books or references that you can recommend?

Start with a Quartz cuvette.

I resuspend my 40nmol oligo stocks in sterile 300ul water or TE (10mM 
Tris pH 8.0, 0.1mM EDTA). Measure 2ul in 1ml water at OD260. From OD 
calculate total no. of OD's in 300ul and then from the calculator 
figures, stock concn. in pM/ul. I would then dilute some of the stock to 
10 or 25pM/ul for use in PCR.

Duncan

-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.


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