Knight:TempliPhi - Revision history

Austin J. Che at 19:43, 18 November 2007

2007-11-18T19:43:39Z

←Older revision		Revision as of 19:43, 18 November 2007
Line 20:		Line 20:
	#Heat to 65°C for 10 mins.		#Heat to 65°C for 10 mins.
	#Cool to 4 °C.		#Cool to 4 °C.
-	#~~Use 1μL to submit~~ for [[Knight:Sequencing DNA\|sequencing]]. ~~<-- ''This is what we used previously when~~ sequencing on the Knight lab sequencer~~.''~~	+	#Send DNA for [[Knight:Sequencing DNA\|sequencing]].
-		+	#* Use 2μL in 12μL for sequencing at the biopolymers facility
		+	#* Previously, for sequencing on the Knight lab sequencer, we used 1μL
	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:In vitro]]		[[Category:In vitro]]
	[[Category:DNA]]		[[Category:DNA]]

Reshma P. Shetty at 18:49, 2 January 2007

2007-01-02T18:49:57Z

←Older revision		Revision as of 18:49, 2 January 2007
Line 6:		Line 6:

	==Procedure==		==Procedure==
-	#Thaw sample buffer (~~white~~ cap) and reaction buffer (blue cap) on ice.	+	#Thaw sample buffer (red cap) and reaction buffer (blue cap) on ice.
	#Transfer 5 μL of sample buffer to small PCR tube for each template to be amplified.		#Transfer 5 μL of sample buffer to small PCR tube for each template to be amplified.
	#Add template to sample buffer		#Add template to sample buffer
Line 14:		Line 14:
	#*Use 1pg-10ng of purified plasmid DNA (volume < 0.5μL)		#*Use 1pg-10ng of purified plasmid DNA (volume < 0.5μL)
	#Heat sample to 95°C for 3 mins to denature and cool to 4°C.		#Heat sample to 95°C for 3 mins to denature and cool to 4°C.
-	#Mix 5 μL reaction buffer with 0.2μL enzyme mix for each reaction. (Make up a master mix for multiple reactions).	+	#Mix 5 μL reaction buffer with 0.2μL enzyme mix for each reaction. (Make up a master mix for multiple reactions.)
	#Transfer 5 μL TempliPhi premix to cooled sample.		#Transfer 5 μL TempliPhi premix to cooled sample.
	#Incubate at 30°C for 4-18 hours.		#Incubate at 30°C for 4-18 hours.

Reshma P. Shetty at 18:32, 2 January 2007

2007-01-02T18:32:21Z

←Older revision		Revision as of 18:32, 2 January 2007
Line 12:		Line 12:
	#*Use small portion of a colony. Avoid transferring any agar.		#*Use small portion of a colony. Avoid transferring any agar.
	#*Dilute 1μL glycerol stock in 50μL water. Use 0.2-0.5 μL.		#*Dilute 1μL glycerol stock in 50μL water. Use 0.2-0.5 μL.
-	#*Use 1pg-10ng of purified plasmid DNA	+	#*Use 1pg-10ng of purified plasmid DNA (volume < 0.5μL)
	#Heat sample to 95°C for 3 mins to denature and cool to 4°C.		#Heat sample to 95°C for 3 mins to denature and cool to 4°C.
	#Mix 5 μL reaction buffer with 0.2μL enzyme mix for each reaction. (Make up a master mix for multiple reactions).		#Mix 5 μL reaction buffer with 0.2μL enzyme mix for each reaction. (Make up a master mix for multiple reactions).

Reshma P. Shetty at 18:01, 2 January 2007

2007-01-02T18:01:38Z

←Older revision		Revision as of 18:01, 2 January 2007
Line 12:		Line 12:
	#*Use small portion of a colony. Avoid transferring any agar.		#*Use small portion of a colony. Avoid transferring any agar.
	#*Dilute 1μL glycerol stock in 50μL water. Use 0.2-0.5 μL.		#*Dilute 1μL glycerol stock in 50μL water. Use 0.2-0.5 μL.
-	#Heat sample to 95°C for 3 mins and cool to 4°C.	+	#*Use 1pg-10ng of purified plasmid DNA
		+	#Heat sample to 95°C for 3 mins to denature and cool to 4°C.
	#Mix 5 μL reaction buffer with 0.2μL enzyme mix for each reaction. (Make up a master mix for multiple reactions).		#Mix 5 μL reaction buffer with 0.2μL enzyme mix for each reaction. (Make up a master mix for multiple reactions).
	#Transfer 5 μL TempliPhi premix to cooled sample.		#Transfer 5 μL TempliPhi premix to cooled sample.

Reshma P. Shetty at 17:16, 2 January 2007

2007-01-02T17:16:09Z

←Older revision		Revision as of 17:16, 2 January 2007
Line 19:		Line 19:
	#Heat to 65°C for 10 mins.		#Heat to 65°C for 10 mins.
	#Cool to 4 °C.		#Cool to 4 °C.
-	#Use 1μL to submit for [[Knight:Sequencing DNA\|sequencing]].	+	#Use 1μL to submit for [[Knight:Sequencing DNA\|sequencing]]. <-- ''This is what we used previously when sequencing on the Knight lab sequencer.''
		+
		+	[[Category:Protocol]]
		+	[[Category:In vitro]]
		+	[[Category:DNA]]

Reshma P. Shetty at 17:09, 2 January 2007

2007-01-02T17:09:44Z

New page

<small>< [[TempliPhi]]</small>

==Materials==
*[http://www6.amershambiosciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&parentid=25640010&moduleid=164891 illustra TempliPhi™ 100/500 Amplification Kit]
* 0.6mL PCR tubes

==Procedure==
#Thaw sample buffer (white cap) and reaction buffer (blue cap) on ice.
#Transfer 5 μL of sample buffer to small PCR tube for each template to be amplified.
#Add template to sample buffer
#*Dilute 1μL saturated overnight culture in 10-100 μL water. Use 0.2-0.5 μL.
#*Use small portion of a colony. Avoid transferring any agar.
#*Dilute 1μL glycerol stock in 50μL water. Use 0.2-0.5 μL.
#Heat sample to 95°C for 3 mins and cool to 4°C.
#Mix 5 μL reaction buffer with 0.2μL enzyme mix for each reaction. (Make up a master mix for multiple reactions).
#Transfer 5 μL TempliPhi premix to cooled sample.
#Incubate at 30°C for 4-18 hours.
#*Incubate overnight for optimal results but 4 hours should be sufficient if there is not too much inhibitory material like agar or rich medium.
#Heat to 65°C for 10 mins.
#Cool to 4 °C.
#Use 1μL to submit for [[Knight:Sequencing DNA|sequencing]].