Knight:In vitro transcription - Revision history

Vaishnavi Ananth at 09:33, 19 November 2009

2009-11-19T09:33:34Z

←Older revision		Revision as of 09:33, 19 November 2009
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	</biblio>		</biblio>

-	==~~BioStream~~ version==	+	==BioCoder version==
-	Following is the Knight:In vitro transcription protocol in ~~BioStream~~, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in ~~BioStream~~ (see Source code). More information about ~~BioStream~~ can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]	+	Following is the Knight:In vitro transcription protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder(see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]
	====Text Output====		====Text Output====
	[[Knight:In vitro transcription protocol]]		[[Knight:In vitro transcription protocol]]

2009-10-09T06:16:29Z

←Older revision		Revision as of 06:16, 9 October 2009
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	#AmbionTemplate [http://www.ambion.com/techlib/tb/tb_154.html A PCR Strategy for Rapid Generation of Template DNA for Synthesis of Labeled RNA Probes] (contains lots of useful info about generating transcription templates via PCR)		#AmbionTemplate [http://www.ambion.com/techlib/tb/tb_154.html A PCR Strategy for Rapid Generation of Template DNA for Synthesis of Labeled RNA Probes] (contains lots of useful info about generating transcription templates via PCR)
	</biblio>		</biblio>
		+
		+	==BioStream version==
		+	Following is the Knight:In vitro transcription protocol in BioStream, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioStream (see Source code). More information about BioStream can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]
		+	====Text Output====
		+	[[Knight:In vitro transcription protocol]]
		+	====Source Code====
		+	[[Knight:In vitro transcription protocol - source code]]

	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:RNA]]		[[Category:RNA]]
	[[Category:In vitro]]		[[Category:In vitro]]

2007-07-19T01:24:52Z

Controls

←Older revision		Revision as of 01:24, 19 July 2007
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	==Controls==		==Controls==
-	There are a few controls that can help to ~~ascerta~~	+	There are a few controls that can help to ascertain that the assay is working properly.

	#Template without RNAP		#Template without RNAP

2007-07-19T01:17:31Z

←Older revision		Revision as of 01:17, 19 July 2007
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	===Analyze transcription products===		===Analyze transcription products===
	Then analyze via [[Knight:RNA electrophoresis/Native\|native agarose gel electrophoresis]].		Then analyze via [[Knight:RNA electrophoresis/Native\|native agarose gel electrophoresis]].
		+
		+	==Controls==
		+	There are a few controls that can help to ascerta
		+
		+	#Template without RNAP
		+	#*To see what just the DNA template looks like when run on a gel.
		+	#Template with RNAP
		+	#*To ensure that the linearized template is transcribed by RNAP.
		+	#Template with RNAP and the buffer that the repressor is resuspended in
		+	#*To verify that any repression seen is not due to altered salt or pH conditions.

	==Notes==		==Notes==

2007-07-19T01:12:03Z

DNase treatment (optional)

←Older revision		Revision as of 01:12, 19 July 2007
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	===DNase treatment (optional)===		===DNase treatment (optional)===
		+	''This step hasn't been tried.''
		+
	An optional step is to treat the reaction with [http://www.neb.com/nebecomm/products/productM0303.asp RNase free DNaseI] to remove the template DNA.		An optional step is to treat the reaction with [http://www.neb.com/nebecomm/products/productM0303.asp RNase free DNaseI] to remove the template DNA.

2007-02-13T22:03:56Z

Option 2: Set up transcription reaction

←Older revision		Revision as of 22:03, 13 February 2007
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	===Option 2: Set up transcription reaction===		===Option 2: Set up transcription reaction===
	#Make up 50 μL reaction		#Make up 50 μL reaction
-	#*22 μL RNase free H<sub>2</sub>O	+	#*25 μL RNase free H<sub>2</sub>O
	#*10 μL 5X ''E. coli'' RNA polymerase transcription buffer		#*10 μL 5X ''E. coli'' RNA polymerase transcription buffer
-	#*~~5 μL of 100 mM DTT <-- replace with~~ 0.5 μL of 500 mM TCEP since DTT chelates zinc	+	#*0.5 μL of 500 mM TCEP (since DTT chelates zinc)
	#*10 μL of 2.5 mM each NTP		#*10 μL of 2.5 mM each NTP
-	#*5 μL of PCR template <-perhaps cut this down? DNA is a pretty bright band?	+	#*2 μL of PCR template <-perhaps cut this down? DNA is a pretty bright band?
	#*2.5 μL ''E. coli'' RNA polymerase holoenzyme		#*2.5 μL ''E. coli'' RNA polymerase holoenzyme
	#Incubate at 37°C for 1 hr.		#Incubate at 37°C for 1 hr.

2007-01-22T15:38:13Z

Option 1: Preincubate repressor and DNA

←Older revision		Revision as of 15:38, 22 January 2007
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	#Incubate 2 hours on benchtop.		#Incubate 2 hours on benchtop.
	#Make up 50 μL reaction		#Make up 50 μL reaction
-	#*25 μL repressor-DNA mixture	+	#*22 μL repressor-DNA mixture
	#*10 μL 5X ''E. coli'' RNA polymerase transcription buffer		#*10 μL 5X ''E. coli'' RNA polymerase transcription buffer
	#*0.5 μL of 500 mM TCEP since DTT chelates zinc		#*0.5 μL of 500 mM TCEP since DTT chelates zinc

2007-01-16T16:59:01Z

Notes

←Older revision		Revision as of 16:59, 16 January 2007
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	*EDTA should be added to a final concentration of 5 mM to protect RNA from being degraded during enzyme inactivation. But EDTA can chelate magnesium which is needed for DNaseI activity. So the EDTA may need to be added just prior to inactivation.		*EDTA should be added to a final concentration of 5 mM to protect RNA from being degraded during enzyme inactivation. But EDTA can chelate magnesium which is needed for DNaseI activity. So the EDTA may need to be added just prior to inactivation.
	*DNase I is not active on DNA bound to proteins.		*DNase I is not active on DNA bound to proteins.
		+	*DTT which is typically included in ''in vitro'' transcription reactions can chelate zinc. Therefore, replace DTT with TCEP when the presence of zinc is necessary.

	===Analyze transcription products===		===Analyze transcription products===

2007-01-16T16:57:11Z

Procedure

←Older revision		Revision as of 16:57, 16 January 2007
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	===Prepare template DNA===		===Prepare template DNA===
-	#Generate linearized template via PCR. Do a 100 μL reaction using VF2 and VR. ~~Use 5 μL directly in the trancription reaction. (Is this enough DNA? Epicentre recommends 1 μg.)~~	+	#Generate linearized template via PCR. Do a 100 μL reaction using VF2 and VR.
	#*''Can be done once, frozen and reused.''		#*''Can be done once, frozen and reused.''

2007-01-16T16:56:31Z

Procedure

←Older revision		Revision as of 16:56, 16 January 2007
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	===Prepare template DNA===		===Prepare template DNA===
	#Generate linearized template via PCR. Do a 100 μL reaction using VF2 and VR. Use 5 μL directly in the trancription reaction. (Is this enough DNA? Epicentre recommends 1 μg.)		#Generate linearized template via PCR. Do a 100 μL reaction using VF2 and VR. Use 5 μL directly in the trancription reaction. (Is this enough DNA? Epicentre recommends 1 μg.)
		+	#*''Can be done once, frozen and reused.''

	===Option 1: Preincubate repressor and DNA===		===Option 1: Preincubate repressor and DNA===