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spectrophotometry determination of DNA

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spectrophotometry determination of DNA

Postby Johnson » Mon Nov 23, 2009 7:50 pm

Please hepl the following;
Given the following;
A solution of 50ug/ml of DNA gives an Absorbance at 260nm of 1. The ratio of absorbance at 260nm and that of 280nm gives an idea of purity. Pure DNA this is 1.8-1.9. Each cell contain 0.005pg of DNA (1ug=10^6pg) and each microfuge tube contain about 1 x 10^2 cells, calculate the concentration of DNA and determine the yield of DNA.
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Postby JackBean » Tue Nov 24, 2009 4:10 am

Wau, what than with samples, where you have A260/A280 more than 2? :lol:
Actually, the pure DNA is for ratio A260/A280 > 1.7

Don't you think, it's a little useless to write 100 as 1x10^2?
If 1 cell contains 0.005pg of DNA, how much DNA will be in 100 cells?

Unless you won't give us your absorbance, we can't calculate the yield ;)
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Postby Johnson » Tue Nov 24, 2009 6:34 am

Ooops! sorry, the A260 was 1.423 and A280 was 0.681.

Thank you
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Postby Johnson » Tue Nov 24, 2009 6:49 am

This is a second problem...

Given 0.026mg (26µg) sample of compound X dissolved in 10.0ml of water. Given also its absorbance
coefficient A^1% of X be 2759, calculate the absorbance, in a 10.0ml cell of 3.0cm path length,
of solution prepared by diluting 3.0ml of the original solution to 10.0ml.

(In the literature, it states, A^1% represents the absorption of a 1% solution of substance
i.e. 1g dissolved in 100ml of solvent. And this has been used because sometimes,
particularly for molecules such as proteins and nucleic acids when it is difficult or impossible
to assign molecular weight, A1% may be quoted at particular wavelength.)

Please need help because am having exam later (three hours from now) today about these topics!!
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Re:

Postby JackBean » Tue Nov 24, 2009 8:42 am

Johnson wrote:Ooops! sorry, the A260 was 1.423 and A280 was 0.681.

You didn't mention the path length, but let's assume, that it's the same as of 50 ug having absorbance 1.
If 50 ug/ml has absorbance 1, what concentration will have absorbance of 1.423 (let's give you advice, that 100 ug/ml have absorbance 2 ;)
I guess to calculate the ratio should not be a problem?
With this you did calculate the concentration, but you need also volume to calculate whole mass of isolated DNA and than you can calculate yield (isolated DNA*100%/teoretical DNA).
http://www.biolib.cz/en/main/

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Re:

Postby JackBean » Tue Nov 24, 2009 8:48 am

Johnson wrote:Given 0.026mg (26µg) sample of compound X dissolved in 10.0ml of water. Given also its absorbance
coefficient A^1% of X be 2759, calculate the absorbance, in a 10.0ml cell of 3.0cm path length,
of solution prepared by diluting 3.0ml of the original solution to 10.0ml.

(In the literature, it states, A^1% represents the absorption of a 1% solution of substance
i.e. 1g dissolved in 100ml of solvent. And this has been used because sometimes,
particularly for molecules such as proteins and nucleic acids when it is difficult or impossible
to assign molecular weight, A1% may be quoted at particular wavelength.)

OK, this looks better now ;)
So, you have basically 0.26mg in 100 ml, that is 2.6*10^-4% solution, right?
Let's assume, you use the same cuvette etc. in both cases ;) So, 1% solution gives you absorbance 2759, what absorbance will have 2.6*10^-4% (let's give you advice, that 0.1% solution will give you absorbance of 275.9;). But! you have diluted it, so the real concentration is (2.6*3/10)*10^-4, OK?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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