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lsl 05-13-2008 11:50 AM

DNA Solution - Some Queries
 
Hello. I hope you can help me to solve some of the problems below.
1) We purchase the synthetic oligonucleotides and they claim to have
100uM of DNA. Is this number reliable for each and every batch?
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)?
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?
Thank you very much for you time.

lsl

Duncan Clark 05-13-2008 12:29 PM

DNA Solution - Some Queries
 
Historians believe that in newspost
<[Only registered and activated users can see links. Click Here To Register...]> on
Tue, 13 May 2008, lsl <[Only registered and activated users can see links. Click Here To Register...]> penned the following literary
masterpiece:

Yield varies batch to batch, oligo to oligo.


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

[Only registered and activated users can see links. Click Here To Register...]

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.


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.

Jose de las Heras 05-13-2008 01:53 PM

DNA Solution - Some Queries
 

"lsl" <[Only registered and activated users can see links. Click Here To Register...]> wrote in message
news:[Only registered and activated users can see links. Click Here To Register...]...

in addition to what Duncan said, don't confuse 100uM (a concentration) with
100umoles (an amount), as the question above seems to imply. I see this too
often in the lab.

Jose



Dr. Hiranya S. Roychowdhury 05-13-2008 06:24 PM

DNA Solution - Some Queries
 
Hmmm ....
In my experience, the molarity is fairly accurate, and certainly is not
generally afactor for routine use of the oligos.

Now, if one has to ask the next two questions, wouldn't it be better that
the downstream apllications are also left up to some commercial outfit?
Should any in the lab have to ask these questions, such a lab is in a
rather bad shape!



--
Hiranya S. Roychowdhury, Ph.D.
Asst. Professor,
Health & Public Services
Dona Ana Community College
New Mexico State University
Las Cruces, NM 88003



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