Weiss Lab:DNA labeling - Revision history

Ron Lin at 00:23, 25 October 2008

2008-10-25T00:23:34Z

←Older revision		Revision as of 00:23, 25 October 2008
Line 1:		Line 1:
	Note: Labeling procedures might be optimized for particular dyes. Check manufacturer instructions.		Note: Labeling procedures might be optimized for particular dyes. Check manufacturer instructions.

-	=Materials=	+	==Materials==

	*Sodium Bicarbonate (pH 8.3, but acceptable range is 8.0 and 8.8): Na(CO3)2		*Sodium Bicarbonate (pH 8.3, but acceptable range is 8.0 and 8.8): Na(CO3)2
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	*TE: 10 mM TRIS (pH 8.0) + 1 mM EDTA		*TE: 10 mM TRIS (pH 8.0) + 1 mM EDTA

-	=Procedure=	+	==Procedure==

	#Suspend DNA from IDT in 100 uL of 50 mM TEAA + 5% ACN		#Suspend DNA from IDT in 100 uL of 50 mM TEAA + 5% ACN
Line 25:		Line 25:
	#Dry in SpeedVacFor long-term storage, leave in TE. Add BSA to prevent sticking if you expect very long-term storage.		#Dry in SpeedVacFor long-term storage, leave in TE. Add BSA to prevent sticking if you expect very long-term storage.

		+	==References==
		+
		+	==Notes==

	[[Category:Protocol]] [[Category:In vitro]] [[Category:DNA]]		[[Category:Protocol]] [[Category:In vitro]] [[Category:DNA]]

Ron Lin at 00:21, 25 October 2008

2008-10-25T00:21:27Z

←Older revision		Revision as of 00:21, 25 October 2008
Line 1:		Line 1:
	Note: Labeling procedures might be optimized for particular dyes. Check manufacturer instructions.		Note: Labeling procedures might be optimized for particular dyes. Check manufacturer instructions.

-	==Materials==	+	=Materials=

	*Sodium Bicarbonate (pH 8.3, but acceptable range is 8.0 and 8.8): Na(CO3)2		*Sodium Bicarbonate (pH 8.3, but acceptable range is 8.0 and 8.8): Na(CO3)2
Line 9:		Line 9:
	*TE: 10 mM TRIS (pH 8.0) + 1 mM EDTA		*TE: 10 mM TRIS (pH 8.0) + 1 mM EDTA

-	==Procedure==	+	=Procedure=

	#Suspend DNA from IDT in 100 uL of 50 mM TEAA + 5% ACN		#Suspend DNA from IDT in 100 uL of 50 mM TEAA + 5% ACN

Ron Lin at 00:21, 25 October 2008

2008-10-25T00:21:01Z

←Older revision		Revision as of 00:21, 25 October 2008
Line 3:		Line 3:
	==Materials==		==Materials==

-	#Sodium Bicarbonate (pH 8.3, but acceptable range is 8.0 and 8.8): Na(CO3)2	+	*Sodium Bicarbonate (pH 8.3, but acceptable range is 8.0 and 8.8): Na(CO3)2
-	#Sodium Borate (pH 8.3)	+	*Sodium Borate (pH 8.3)
-	#Acetonitrile: ACN	+	*Acetonitrile: ACN (HPLC grade)
-	#Sodium Acetate (pH 5.2)	+	*Sodium Acetate (pH 5.2)
-	#TE: 10 mM TRIS (pH 8.0) + 1 mM EDTA	+	*TE: 10 mM TRIS (pH 8.0) + 1 mM EDTA

	==Procedure==		==Procedure==
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	#HPLC and collect unlabeled peak, labeled peak		#HPLC and collect unlabeled peak, labeled peak
	#Dry in SpeedVacFor long-term storage, leave in TE. Add BSA to prevent sticking if you expect very long-term storage.		#Dry in SpeedVacFor long-term storage, leave in TE. Add BSA to prevent sticking if you expect very long-term storage.
		+
		+
		+	[[Category:Protocol]] [[Category:In vitro]] [[Category:DNA]]

Ron Lin at 00:19, 25 October 2008

2008-10-25T00:19:54Z

←Older revision		Revision as of 00:19, 25 October 2008
Line 1:		Line 1:
-	Note: Labeling procedures might be optimized for particular dyes. Check manufacturer instructions.	+	Note: Labeling procedures might be optimized for particular dyes. Check manufacturer instructions.
-	~~Materials:~~	+

	==Materials==		==Materials==

-	Sodium Bicarbonate (pH 8.3, but acceptable range is 8.0 and 8.8): Na(CO3)2	+	#Sodium Bicarbonate (pH 8.3, but acceptable range is 8.0 and 8.8): Na(CO3)2
-	Sodium Borate (pH 8.3)	+	#Sodium Borate (pH 8.3)
-	Acetonitrile: ACN	+	#Acetonitrile: ACN
-	Sodium Acetate (pH 5.2)	+	#Sodium Acetate (pH 5.2)
-	TE: 10 mM TRIS (pH 8.0) + 1 mM EDTA	+	#TE: 10 mM TRIS (pH 8.0) + 1 mM EDTA

	==Procedure==		==Procedure==

Ron Lin: New page: Note: Labeling procedures might be optimized for particular dyes. Check manufacturer instructions. Materials: ==Materials== Sodium Bicarbonate (pH 8.3, but acceptable range is 8.0 and ...

2008-10-25T00:18:24Z

New page: Note: Labeling procedures might be optimized for particular dyes. Check manufacturer instructions. Materials: ==Materials== Sodium Bicarbonate (pH 8.3, but acceptable range is 8.0 and ...

New page

Note: Labeling procedures might be optimized for particular dyes. Check manufacturer instructions.
Materials:

==Materials==

Sodium Bicarbonate (pH 8.3, but acceptable range is 8.0 and 8.8): Na(CO3)2
Sodium Borate (pH 8.3)
Acetonitrile: ACN
Sodium Acetate (pH 5.2)
TE: 10 mM TRIS (pH 8.0) + 1 mM EDTA

==Procedure==

#Suspend DNA from IDT in 100 uL of 50 mM TEAA + 5% ACN
##To help dissolve, raise the temperature for 10 mins if necessary
#Spin down, pellet will form powdery-crystal residue which is the particulate from the sample that is not DNA. Keep it just in case, since there might be some DNA in that pellet.
#HPLC to separate lengths (use Reverse Phase/Size Exclusion column)
##There will be one large fraction which is the fraction of interest
#Dry the elution/fraction in SpeedVac. After drying, suspend dried fraction in MilliQ water. Check absorbance to determine concentration in UV/Vis Spectrometer.
#For labeling the complement strand with ATTO 647, resuspend 100 mM (pH 8.3) Sodium Bicarbonate. For labeling template strand with Cy3B, use 100 mM (pH 8.3) Sodium Borate. Determine concentration of dyes in DMSO using absorbance.
Add dye in excess (10x recommended). Dissolve the dye in anhydrous DMSO/DMF, prepared immediately before labeling reaction. Leave for 24-48 hrs at room temperature.
#Ethanol Precipitate. Add 150 mM Sodium Acetate and 2x volume of ethanol
##For a 100 uL solution, add 5 uL of Sodium Acetate and 250 uL of ethanol
Place in (-80 or -20) freezer at least 1 hr, overnight is fine. Remove supernatant and make sure all ethanol has evaporated off. #Resuspend in 50 mM TEAA + 5% ACN
#HPLC and collect unlabeled peak, labeled peak
#Dry in SpeedVacFor long-term storage, leave in TE. Add BSA to prevent sticking if you expect very long-term storage.