Sauer:Nucleic acid quantitation - Revision history

Smoore at 19:06, 5 February 2007

2007-02-05T19:06:49Z

←Older revision		Revision as of 19:06, 5 February 2007
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	[http://paris.chem.yale.edu/extinct.html Extinction coefficient and Tm calculator]		[http://paris.chem.yale.edu/extinct.html Extinction coefficient and Tm calculator]

-	~~The originator actually meant "quantification", sorry. (Actually. quantitation is also correct. The definition for both~~ of the ~~verbs "quantitate" and "quantify" is "to determine the quantity~~ of~~", with quantitation and quantification being~~ the ~~noun form of each verb, respectively. According to a quick google search, both are commonly used when referring to measuring the concentration of nucleic acids. The~~ words ~~"precision"~~ and ~~"measure" only occur in~~ the "~~quantitate~~" ~~definition, and not the "quantify" definition at [http://dictionary.reference.com/ dictionary.com], which may make this the more accurate choice, but it seems like both are acceptable~~.~~--[[User:Kathmc\|Kathleen]])~~	+	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].

Kathmc at 00:10, 26 January 2007

2007-01-26T00:10:15Z

←Older revision		Revision as of 00:10, 26 January 2007
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	[http://paris.chem.yale.edu/extinct.html Extinction coefficient and Tm calculator]		[http://paris.chem.yale.edu/extinct.html Extinction coefficient and Tm calculator]

-	The originator actually meant "quantification", sorry.	+	The originator actually meant "quantification", sorry. (Actually. quantitation is also correct. The definition for both of the verbs "quantitate" and "quantify" is "to determine the quantity of", with quantitation and quantification being the noun form of each verb, respectively. According to a quick google search, both are commonly used when referring to measuring the concentration of nucleic acids. The words "precision" and "measure" only occur in the "quantitate" definition, and not the "quantify" definition at [http://dictionary.reference.com/ dictionary.com], which may make this the more accurate choice, but it seems like both are acceptable.--[[User:Kathmc\|Kathleen]])

	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].

Smoore at 23:26, 25 January 2007

2007-01-25T23:26:25Z

←Older revision		Revision as of 23:26, 25 January 2007
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	[http://paris.chem.yale.edu/extinct.html Extinction coefficient and Tm calculator]		[http://paris.chem.yale.edu/extinct.html Extinction coefficient and Tm calculator]

-	The originator actually ~~mean~~ "quantification" sorry.	+	The originator actually meant "quantification", sorry.

	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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	===RNA===		===RNA===

-	ss RNA, Divide by 0.025. There is no good way of calculating a value for folded RNA so see the next entry.	+	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===		===Making your own RNA Extinction coefficients===

Smoore at 23:23, 25 January 2007

2007-01-25T23:23:40Z

←Older revision		Revision as of 23:23, 25 January 2007
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	{{Sauer lab sidebar}}		{{Sauer lab sidebar}}
		+	contributed by [[Sean Moore]]
		+
		+	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]

Smoore: /* Making your own RNA Extinction coefficients */

2007-01-25T23:19:54Z

Making your own RNA Extinction coefficients

←Older revision		Revision as of 23:19, 25 January 2007
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	70S = 40,453,067		70S = 40,453,067

-	I usually dilute a ribosome prep 1/500 to 1/1000 in PO<sub>4</sub> buffer and measure that.	+
		+	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.

Smoore: /* Making your own RNA Extinction coefficients */

2007-01-25T23:18:39Z

Making your own RNA Extinction coefficients

←Older revision		Revision as of 23:18, 25 January 2007
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	70S = 39,103,438		70S = 39,103,438

-	<b>In 100 mM K<sup>+</sup>glutamate, 6 mM MgCl<~~suv~~>2</sub>, 20 mM Tris-HEPES pH 7.8, 0.05% Tween-20, 14 mM mercaptoethanol</b>	+	<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>

	50S = 27,058,100		50S = 27,058,100

Smoore: /* Making your own RNA Extinction coefficients */

2007-01-25T23:18:06Z

Making your own RNA Extinction coefficients

←Older revision		Revision as of 23:18, 25 January 2007
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	Using the observed A<sub>260</sub> for your fully hydrolyzed RNA sample, determine the concentration of your RNA.		Using the observed A<sub>260</sub> for your fully hydrolyzed RNA sample, determine the concentration of your RNA.

-	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 ~~wont~~ have to hydrolyze the samples, just dilute them in a buffer an measure.	+	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.

-	I calculated the following:	+	I calculated the following for E. coli ribosomal subunit RNAs:
		+
		+	<b>Hydrolyzed:</B>
		+
		+	23S = 33,386,832
		+
		+	16S = 17,588,970
		+
		+	5S = 1,318,308
		+
		+	23S + 5S (large subunit) = 34,705,140
		+
		+	<b>In 100mM NaH<sub>X</sub>PO<sub>4</sub> 1 mM EDTA:</b>
		+
		+	50S = 25,457,162
		+
		+	30S = 13,646,276
		+
		+	70S = 39,103,438
		+
		+	<b>In 100 mM K<sup>+</sup>glutamate, 6 mM MgCl<suv>2</sub>, 20 mM Tris-HEPES pH 7.8, 0.05% Tween-20, 14 mM mercaptoethanol</b>
		+
		+	50S = 27,058,100
		+
		+	30S = 13,394,967
		+
		+	70S = 40,453,067
		+
		+	I usually dilute a ribosome prep 1/500 to 1/1000 in PO<sub>4</sub> buffer and measure that.

Smoore: /* Making your own Extinction coefficients */

2007-01-25T23:07:55Z

Making your own Extinction coefficients

←Older revision		Revision as of 23:07, 25 January 2007
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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.		ss RNA, Divide by 0.025. There is no good way of calculating a value for folded RNA so see the next entry.

-	===Making your own Extinction coefficients===	+	===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 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).
		+
		+	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 A<sub>260</sub> for your fully hydrolyzed RNA sample, determine the concentration of your RNA.
		+
		+	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 wont have to hydrolyze the samples, just dilute them in a buffer an measure.
		+
		+	I calculated the following:

Smoore at 22:53, 25 January 2007

2007-01-25T22:53:14Z

←Older revision		Revision as of 22:53, 25 January 2007
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	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].
		+
		+	===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.
		+
		+	===Making your own Extinction coefficients===

Smoore at 22:45, 25 January 2007

2007-01-25T22:45:43Z

←Older revision		Revision as of 22:45, 25 January 2007
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	{{Sauer lab sidebar}}		{{Sauer lab sidebar}}
	[http://paris.chem.yale.edu/extinct.html Extinction coefficient and Tm calculator]		[http://paris.chem.yale.edu/extinct.html Extinction coefficient and Tm calculator]
		+
		+	The originator actually mean "quantification" sorry.

	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].