Prbbbb:dna measurement on plate v1 - Revision history

Dan T.A. Eisenberg at 03:20, 7 January 2011

2011-01-07T03:20:53Z

←Older revision		Revision as of 03:20, 7 January 2011
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	*384 well UV transmissive plate with flat clear bottom		*384 well UV transmissive plate with flat clear bottom
	** we use Greiner UV star Cat # 781 801		** we use Greiner UV star Cat # 781 801
		+	*Greiner 788876 are also recommended for even lower volumes (although they are kind of expensive, about $10/plate and only come in packs of 80)
	* miniprepped DNA, at least 30 µl		* miniprepped DNA, at least 30 µl

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	of each well, we can easily calculate the path length for each volume. At least with the Greiner plate we		of each well, we can easily calculate the path length for each volume. At least with the Greiner plate we
	tried, this works surprisingly well and there is no need for calibration curves.		tried, this works surprisingly well and there is no need for calibration curves.
		+	You can also calculate the pathlength empirically on a well-by-well basis by getting the absorption at 977 and 900 and then using the equation. This techniques may be superior because it should partially control for pippeting error as well as sample adhering to the edges of the wells.
		+	<math> d=(A977-A900)/0.18
		+	</math>

	'''parameters'''		'''parameters'''
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	# Pipette exact volume of DNA samples into 384 well plate		# Pipette exact volume of DNA samples into 384 well plate
-	#* multi-channel pipetting from a 96-well plate works	+	#* multi-channel pipetting from a 96-well plate works (SABiosciences PA-384 guides help when x-fering between 96 and 384 well plates)
	#* note, the pipetting is the biggest source of error		#* note, the pipetting is the biggest source of error
	# measure 260 nm and 280 nm absorbance on plate reader		# measure 260 nm and 280 nm absorbance on plate reader

Dan T.A. Eisenberg: /* Overview */

2011-01-07T03:14:11Z

Overview

←Older revision		Revision as of 03:14, 7 January 2011
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	'''Limits'''		'''Limits'''
	Concentrations down to 10 ng/µl can be reliably measured. The volume should be 20 µl or more per well. The samples are later recovered for dilution and storage.		Concentrations down to 10 ng/µl can be reliably measured. The volume should be 20 µl or more per well. The samples are later recovered for dilution and storage.
		+	Using Greiner 788876 plates, I have had good luck down to 10 ul (DTAE).

	==Materials==		==Materials==

Raik: /* Notes */

2009-05-28T21:34:35Z

Notes

←Older revision		Revision as of 21:34, 28 May 2009
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	<font face="courier"><nowiki>raik:</nowiki></font>		<font face="courier"><nowiki>raik:</nowiki></font>
-	~~This~~ protocol ~~is quick and robust in my hands.~~	+	We are just starting to use this protocol -- share your experience!

	<!--Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.-->		<!--Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.-->

Raik: /* Procedure */

2009-05-28T21:33:24Z

Procedure

←Older revision		Revision as of 21:33, 28 May 2009
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	# Pipette exact volume of DNA samples into 384 well plate		# Pipette exact volume of DNA samples into 384 well plate
-	* multi-channel pipetting from a 96-well plate works	+	#* multi-channel pipetting from a 96-well plate works
-	* note, the pipetting is the biggest source of error	+	#* note, the pipetting is the biggest source of error
	# measure 260 nm and 280 nm absorbance on plate reader		# measure 260 nm and 280 nm absorbance on plate reader
	# Pipette sample back into storage plate		# Pipette sample back into storage plate

Raik: /* Procedure */

2009-05-28T21:33:02Z

Procedure

←Older revision		Revision as of 21:33, 28 May 2009
Line 62:		Line 62:

	# Pipette exact volume of DNA samples into 384 well plate		# Pipette exact volume of DNA samples into 384 well plate
-	** multi-channel pipetting from a 96-well plate works	+	* multi-channel pipetting from a 96-well plate works
-	** note, the pipetting is the biggest source of error	+	* note, the pipetting is the biggest source of error
	# measure 260 nm and 280 nm absorbance on plate reader		# measure 260 nm and 280 nm absorbance on plate reader
	# Pipette sample back into storage plate		# Pipette sample back into storage plate

Raik: /* Procedure */

2009-05-28T21:32:39Z

Procedure

←Older revision		Revision as of 21:32, 28 May 2009
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	# Pipette exact volume of DNA samples into 384 well plate		# Pipette exact volume of DNA samples into 384 well plate
		+	** multi-channel pipetting from a 96-well plate works
		+	** note, the pipetting is the biggest source of error
	# measure 260 nm and 280 nm absorbance on plate reader		# measure 260 nm and 280 nm absorbance on plate reader
	# Pipette sample back into storage plate		# Pipette sample back into storage plate

Raik: /* Calculation */

2009-05-28T21:23:41Z

Calculation

←Older revision		Revision as of 21:23, 28 May 2009
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	<math>		<math>
-	c = \frac{(A-A0) \~~dot~~ e}{d}	+	c = \frac{(A-A0) \cdot e}{d}
	</math>		</math>

Raik: /* Calculation */

2009-05-28T21:22:46Z

Calculation

←Older revision		Revision as of 21:22, 28 May 2009
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	this path length depends on how much sample you fill into your wells. If we know the exact inner dimensions		this path length depends on how much sample you fill into your wells. If we know the exact inner dimensions
	of each well, we can easily calculate the path length for each volume. At least with the Greiner plate we		of each well, we can easily calculate the path length for each volume. At least with the Greiner plate we
-	tried, this works surprisingly well.	+	tried, this works surprisingly well and there is no need for calibration curves.

	'''parameters'''		'''parameters'''
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	'''formula'''		'''formula'''
		+
		+	Once the path length d (in cm) is known, the DNA concentration is calculated from the measured absorption A
		+	(corrected by the blank absorption A0):

	<math>		<math>
-	c = \frac{(A-A0) \~~times~~ e}{d}	+	c = \frac{(A-A0) \dot e}{d}
	</math>		</math>

Raik: /* Calculation */

2009-05-28T21:19:11Z

Calculation

←Older revision		Revision as of 21:19, 28 May 2009
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	==Calculation==		==Calculation==
		+
		+	Absorption is proportional to the path length through your sample. On a microtiter plate,
		+	this path length depends on how much sample you fill into your wells. If we know the exact inner dimensions
		+	of each well, we can easily calculate the path length for each volume. At least with the Greiner plate we
		+	tried, this works surprisingly well.

	'''parameters'''		'''parameters'''

-	* well dimension: flat bottom 3.3 mm x 3.3 mm
	* DNA extinction coefficient e: 50 [(ng * cm) / µl]		* DNA extinction coefficient e: 50 [(ng * cm) / µl]
		+
		+	* well dimension: flat bottom 3.3 mm x 3.3 mm

	* path length in mm (d') or in cm (d):		* path length in mm (d') or in cm (d):

Raik: /* Calculation */

2009-05-28T21:11:57Z

Calculation

←Older revision		Revision as of 21:11, 28 May 2009
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	* A0...Absorbance of blank sample (same amount, same buffer/water)		* A0...Absorbance of blank sample (same amount, same buffer/water)
	* d... path length in cm		* d... path length in cm
-	* e... extinction coefficient in ~~<math>~~ng * cm / µl~~</math>~~	+	* e... extinction coefficient in ng*cm/µl

	==Procedure==		==Procedure==