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need some help

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3 replies to this topic

#1 claritylight



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Posted 23 July 2009 - 09:31 AM

Say I have a stock concentration of DNA which is assumed to be 2000ng/ul. I take 2.5ul of this into 197.5ul of 1x TE, to make 200ul of 5ug final working stock, final concentration is 0.025ug/ul or 25 ng/ul.

Then I do Picogreen assay (it is like nanodrop, but more sensitive to dsDNA), taking 1ul out of 200ul of the 5ug working stock. The reading is 130 ng/mL.

Does this reading mean that I have 0.130ng/ul as measured by picogreen & how do I calculate back to the number of micrograms I have based on this reading?

Edited by claritylight, 23 July 2009 - 11:41 AM.

#2 georgiadave



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Posted 23 July 2009 - 03:07 PM

Your calculations are correct, I too get 25ng/ul. I do not understand why your reading is so low, maybe a pipetting error?

To estimate the total number of micrograms in your solution multiply 0.130ng/ul * 200ul = 26ng total.

Do you have a tube of DNA that you know the concentration of to determine if it is a machine error?

#3 little mouse

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Posted 24 July 2009 - 12:17 AM

stupid question, but when you took 1 ÁL of your 25 ng/ÁL solution to quantify it : you diluted it, don't you?
how much did you dilute it? (200 times ?)

if this is right, you just forgot to take it into account ?

#4 almost a doctor

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Posted 27 July 2009 - 06:05 AM

Agree with Little Mouse, if I remember correctly how PicoGreen works, you'll have add that 1ul of your DNA to some TE and PicoGreen reagent... total 200ul... so taking this dilution into account, your concentration calculation by PicoGreen, is pretty good!

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