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question on calculation DNA amount


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#1 lyok

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Posted 29 July 2014 - 10:51 AM

Dear all,

 
I am having a problem with a calculation of the amount of oligos in my sample.
 
 
I ordered oligos from IDT and I got back the following information on the paper:
 
Amount of oligo:
OD260= 6,4 
= 23,5 nMoles
= 0,20 mg 
 
If I read this, I understand it as follows: the sample contains 0,20mg of DNA (oligo).
Now what I do not understand is the following:
 
I dissolve the oligo in 235 micro liters.
(= 23,5 x 10) 
 
So I would think: I have 0,20 mg in 235 microliters….
 
When you have 0,20 mg in total and you dissolve it in 235 microliters you would thus have: 0,20 mg/235 microliter
or  0,20/235 = 0,000851 mg per microliter or 0,851 micrograms per microliter or 851 nano grams per microliter
This is however not what I measured with the nano drop 
 
When I measure the amount of DNA with the nano drop I get a result of +- 1300 nano grams / microliter…
 
 
How can dissolving 0,20 mg in 235 microliter end up in 1300 ng/microliter???
 
Am I doing something wrong here or is it safe to say that the nanodrop is just messed up completely.
 
 


#2 Trof

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Posted 29 July 2014 - 12:31 PM

The calculation is right.

I would trust IDT more that I would ever trust Nanodrop.

 

If you don't mixed up the volume. What means 23,5 x 10? You pipetted 23,5 ul ten times??

 

There may be something that increased the measured concentration, either in the sample or in the optical path.


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#3 lyok

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Posted 30 July 2014 - 01:12 AM

I just ment I added 235 microliters (10 times the nanomolar concentration).

 

Ok, I knew it had to be correct but because of the weird nanodrop measurements I started doubting myself.



#4 mdfenko

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Posted 30 July 2014 - 03:32 AM

there may be residual buffer salts with the oligo that are increasing the reading.


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#5 Trof

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Posted 30 July 2014 - 04:20 AM

But that is unlikely in commercial oligos. Desalting is the standard purification procedure.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#6 mlomonaco

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Posted 30 July 2014 - 06:47 AM

The A260 by nanodrop should be arithmetically identical to the value supplied by IDT.  IDT determines mass from the extinction coefficient of the oligo.  Nanodrop determines mass without regard to base composition, therefore it is not reliable for oligos because there is no allowance ACGT.






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