Sauer:Nucleic acid quantitation

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contributed by [[Sean Moore]]
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this link has been dead for some time now:
[http://paris.chem.yale.edu/extinct.html Extinction coefficient and Tm calculator]
[http://paris.chem.yale.edu/extinct.html Extinction coefficient and Tm calculator]
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The originator actually mean "quantification" sorry.
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See a discussion of the use of the words quantification and quantitation on the "Discussion" page.
Ambion has a good site that gives a variety of parameters for nucleotides and nucleic acids [http://www.ambion.com/techlib/append/na_mw_tables.html].
Ambion has a good site that gives a variety of parameters for nucleotides and nucleic acids [http://www.ambion.com/techlib/append/na_mw_tables.html].
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===Intro===
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Lot's of ways.  I like the spec.
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===Measuring nucleic acid concentration in a spectrophotometer===
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Pipette your nucleic acid sample into a cuvette and record an absorbance scan from 240-340 nm.
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Most calculations are done from the Abs at 260 nm.
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====DNA====
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Divide the absorbance at 260 nm by 0.02 for dsDNA or 0.027 for ssDNA.  This is the "concentration" in μg/mL.  Actually, to get concentration, you have to know the length of your molecule and use the mass/volume to calculate a concentration.
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===RNA===
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ss RNA, Divide by 0.025.  There is no good way of calculating a value for folded RNA so see the next entry.  You'll notice that the peak of absorbance for RNA is left of 260, this is normal.
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===Making your own RNA Extinction coefficients===
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I couldn't find extinction coefficient for individual ribosomal subunits, so I did the following.
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Use this reference for more info:  Cavaluzzi MJ, Borer PN. 2004.
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I purified the subunits.  Then, I prepared several samples.  Each was diluted to the same extent in the final reaction.  One "Native" folded form that was in a translation buffer, one "dissociated" in Phosphate and EDTA, and one "hydrolysed" to get an accurate concentration measurement.
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The hydrolyzed sample was prepared by incubating 5 &mu;L of ribosome with 20 &mu;L of 1.0 N NaOH for 1 hour at 37 degrees.  Then I neutralized the sample with 20 &mu;L of 1.0 N HCl.  I Added 1 mL of 100 mM NaH<sub>X</sub>PO<sub>4</sub> at pH 7.0 with 1 mM EDTA.  (this is the same pH used by Cavaluzzi and Borer).
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Measure the absorbance of all three samples.  I did mine in triplicate and averaged the final values.
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So, use the known extinction coefficients obtained by Cavaluzzi and Borer.  Use a word processor to determine the number of each nucleotide in your particular RNA and multiply by the extinction coefficient for that nucleotide.  Then sum the four values.  This will give a "formal" extinction coefficient for your RNA. 
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Ext. Coeffs at 260;
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pA = 15,020
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pC = 7,070
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pG = 12,808
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pU = 9,660
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Using the observed A<sub>260</sub> for your fully hydrolyzed RNA sample, determine the concentration of your RNA.
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Now, with that known concentration and the measured A<sub>260</sub> of the native and unfolded samples, generate extinction coefficients for these conditions.  In the future, you won't have to hydrolyze the samples, just dilute them in a buffer an measure.
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I calculated the following for E. coli ribosomal subunit RNAs:
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<b>Hydrolyzed:</B>
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23S = 33,386,832
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16S = 17,588,970
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5S  = 1,318,308
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23S + 5S (large subunit) = 34,705,140
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<b>In 100mM NaH<sub>X</sub>PO<sub>4</sub> 1 mM EDTA:</b>
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50S = 25,457,162
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30S = 13,646,276
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70S = 39,103,438
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<b>In 100 mM K<sup>+</sup>glutamate, 6 mM MgCl<sub>2</sub>, 20 mM Tris-HEPES pH 7.8, 0.05% Tween-20, 14 mM mercaptoethanol</b>
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50S = 27,058,100
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30S = 13,394,967
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70S = 40,453,067
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I usually dilute a ribosome prep 1/500 to 1/1000 in PO<sub>4</sub> buffer and measure that because the tween and meercaptoethanol absorb like crazy and make variable baselines.

Current revision

SAUER LAB

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contributed by Sean Moore

this link has been dead for some time now: Extinction coefficient and Tm calculator

See a discussion of the use of the words quantification and quantitation on the "Discussion" page.

Ambion has a good site that gives a variety of parameters for nucleotides and nucleic acids [1].

Contents

Intro

Lot's of ways. I like the spec.


Measuring nucleic acid concentration in a spectrophotometer

Pipette your nucleic acid sample into a cuvette and record an absorbance scan from 240-340 nm.

Most calculations are done from the Abs at 260 nm.

DNA

Divide the absorbance at 260 nm by 0.02 for dsDNA or 0.027 for ssDNA. This is the "concentration" in μg/mL. Actually, to get concentration, you have to know the length of your molecule and use the mass/volume to calculate a concentration.

RNA

ss RNA, Divide by 0.025. There is no good way of calculating a value for folded RNA so see the next entry. You'll notice that the peak of absorbance for RNA is left of 260, this is normal.

Making your own RNA Extinction coefficients

I couldn't find extinction coefficient for individual ribosomal subunits, so I did the following.

Use this reference for more info: Cavaluzzi MJ, Borer PN. 2004.

I purified the subunits. Then, I prepared several samples. Each was diluted to the same extent in the final reaction. One "Native" folded form that was in a translation buffer, one "dissociated" in Phosphate and EDTA, and one "hydrolysed" to get an accurate concentration measurement.

The hydrolyzed sample was prepared by incubating 5 μL of ribosome with 20 μL of 1.0 N NaOH for 1 hour at 37 degrees. Then I neutralized the sample with 20 μL of 1.0 N HCl. I Added 1 mL of 100 mM NaHXPO4 at pH 7.0 with 1 mM EDTA. (this is the same pH used by Cavaluzzi and Borer).

Measure the absorbance of all three samples. I did mine in triplicate and averaged the final values.

So, use the known extinction coefficients obtained by Cavaluzzi and Borer. Use a word processor to determine the number of each nucleotide in your particular RNA and multiply by the extinction coefficient for that nucleotide. Then sum the four values. This will give a "formal" extinction coefficient for your RNA.

Ext. Coeffs at 260;

pA = 15,020

pC = 7,070

pG = 12,808

pU = 9,660


Using the observed A260 for your fully hydrolyzed RNA sample, determine the concentration of your RNA.

Now, with that known concentration and the measured A260 of the native and unfolded samples, generate extinction coefficients for these conditions. In the future, you won't have to hydrolyze the samples, just dilute them in a buffer an measure.

I calculated the following for E. coli ribosomal subunit RNAs:

Hydrolyzed:

23S = 33,386,832

16S = 17,588,970

5S = 1,318,308

23S + 5S (large subunit) = 34,705,140

In 100mM NaHXPO4 1 mM EDTA:

50S = 25,457,162

30S = 13,646,276

70S = 39,103,438

In 100 mM K+glutamate, 6 mM MgCl2, 20 mM Tris-HEPES pH 7.8, 0.05% Tween-20, 14 mM mercaptoethanol

50S = 27,058,100

30S = 13,394,967

70S = 40,453,067


I usually dilute a ribosome prep 1/500 to 1/1000 in PO4 buffer and measure that because the tween and meercaptoethanol absorb like crazy and make variable baselines.

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