Reshma P. Shetty at 16:06, 12 December 2006

2006-12-12T16:06:45Z

←Older revision		Revision as of 16:06, 12 December 2006
Line 1:		Line 1:
	==Other reaction conditions from the literature==		==Other reaction conditions from the literature==
		+
		+	===Epicentre===
		+	From Epicentre.
		+
		+	====Functional test====
		+	50 μL reaction
		+	*1X ''E. coli'' RNA polymerase transcription buffer
		+	**0.04 M Tris-HCl (pH 7.5)
		+	**0.15 M KCl
		+	**10 mM MgCl<sub>2</sub>
		+	**0.01% Triton® X-100
		+	*2mM DTT
		+	*0.25mM NTPs
		+	*1μg template
		+
		+	====Assay test====
		+	50 μL reaction
		+	*1X ''E. coli'' RNA polymerase transcription buffer
		+	**0.04 M Tris-HCl (pH 7.5)
		+	**0.15 M KCl
		+	**10 mM MgCl<sub>2</sub>
		+	**0.01% Triton® X-100
		+	*10 mM DTT
		+	*0.5 mM NTPs
		+	*1μg template
		+

	===Szalewska-Palasz et al.===		===Szalewska-Palasz et al.===

Reshma P. Shetty: /* Galan et al */

2006-12-12T16:04:25Z

Galan et al

←Older revision		Revision as of 16:04, 12 December 2006
Line 46:		Line 46:
	</biblio>		</biblio>

-	===Galan et al===	+	===Galan et al.===
	(Run off transcription assay)		(Run off transcription assay)

Reshma P. Shetty: /* Vo et al. */

2006-12-12T16:00:24Z

Vo et al.

←Older revision		Revision as of 16:00, 12 December 2006
Line 124:		Line 124:
	*150 mM KCl		*150 mM KCl
	*100μM each of four NTPs		*100μM each of four NTPs
-	*[γ-<~~sup32~~</sup>P]ATP (~5cpm/fmol) in buffer	+	*[γ-<sup>32</sup>P]ATP (~5cpm/fmol) in buffer
	**40 mM Tris-HCl pH8		**40 mM Tris-HCl pH8
	**10 mM MgCl<sub>2</sub>		**10 mM MgCl<sub>2</sub>

Reshma P. Shetty at 01:45, 7 December 2006

2006-12-07T01:45:55Z

New page

==Other reaction conditions from the literature==

===Szalewska-Palasz et al.===
====25 μL reaction====
*Buffer M
**20 mM Hepes, pH 8.0
**5 mM magnesium acetate
**4 mM DTT
**1 mM EDTA
**1mM ATP
**BSA (5mg/mL)
**0.2% Triton X-100
**5% glycerol
*5 μg template DNA

====Procedure====
#Add repressor
#Incubate 5min at 37°C
#Transfer samples to ice bath
#Add 1 unit ''E. coli'' RNAP from Epicentre
#Add 150 μM CTP and GTP
#Add 1 mM ATP
#Add 15 μM UTP
#Add [α-32P]UTP to 1mCi/mL
#Incubate samples at 37°C for 12.5 mins
#Stop reaction with equal volume of
#*BSA (1.2mg/mL)
#*0.1 mM EDTA, pH 8.0
#*5.1 M ammonium acetate
#Transfer to ice bath
#Precipitate RNA with 2 volumes 96% ethanol in liquid nitrogen
#Centrifuge in microcentrifuge at maximum speed for 30 mins
#Dry pellet
#Resuspend in 20 μL of
#*98% formamide
#*0.25% bromophenol blue
#*0.25% xylene cyanol
#Incubate at 65°C for 5 mins
#Electrophorese in 6% polyacrylamide gel containing 46% urea in TBE buffer at 30mA
#Dry the gel
#Visualize RNA bands by autoradiography and quantify via densitometry

====Reference====
<biblio>
#Szalewska-Palasz-PNAS-1998 pmid=9539721
</biblio>

===Galan et al===
(Run off transcription assay)

====9 μL reaction====
*5nM DNA (supercoiled plasmid)
*100nM CRP
*100nM HpaR or buffer B

*Buffer B
**40 mM Tris-HCl pH 8.0
**10 mM MgCl2
**100 mM KCl
**200 μM cAMP
**500 μg/mL acetylated BSA

====Procedure====
#Incubate reaction at room temperature for 20 mins
#Add 3 μL RNAp at 375 nM in buffer B
#Incubate 5 mins at 37°C
#Start elongation with 3 μL of prewarmed mixture in buffer B of
#*1mM ATP
#*1mM GTP
#*1mM CTP
#*50μM UTP
#*1μCi [α-32]UTP
#*500 μg/mL heparin
#Incubate 5 mins at 37°C
#Add 12μL loading buffer containing 1% SDS
#Heat to 70°C
#Electrophorese samples on 7% sequencing gels
#Quantify using phosphorimager

====Reference====
<biblio>
#Galan-NAR-2003 pmid=14602920
</biblio>

===Marschall et al.===
Run off transcription assay

#Used either supercoiled or linear template
#5 μL of 20nM DNA template in potassium glutamate solution
#*40 mM Hepes (pH 8.0)
#*10 mM magnesium chloride
#*100 mM potassium glutamate
#*200 μM cAMP
#*500 μg/mL acetylated bovine serum albumin
#Add 5 μL of 400 nM Lrp or buffer
#Add 5 μL of 400 nM Crp or buffer
#Incubate at room temperature for 15 mins
#7μL of mixture was incubated at 37°C for 5 mins
#Add 3.5 μL RNAp at 260 nM
#Incubate for 5 mins at 37°C
#Start polymerization by adding 3.5 μL prewarmed mixture containing
#*1mM ATP
#*1mM GTP
#*1mM CTP
#*50μM UTP
#*500 μg/mL heparin
#*1μCi [α-32]UTP
#Incubate 5 mins (what temp?)
#Stop reaction by adding 20mM EDTA in formamide containing xylene cyanol and bromophenol blue
#Heat to 65°C
#Electrophorese samples on 7% sequencing gels
#Autoradiograph and quantify using phosphorimager

====Reference====
<biblio>
#Marschall-JMB-1998 pmid=9512707
</biblio>

===Vo et al.===
Steady state transcription assay

20-50μL reaction mixtures (prepped on ice)
*30 nM promoter template
*150 mM KCl
*100μM each of four NTPs
*[γ-<sup32P]ATP (~5cpm/fmol) in buffer
**40 mM Tris-HCl pH8
**10 mM MgCl2
**10 mM β-mercaptoethanol
**10 μg/mL acetylated BSA
#Add E. coli RNA polymerase to final concentration of 30 nM
#Incubate at 37°C
#Remove 5-10 μL samples at 0-60 mins
#Mix with equal volume of formamide loading buffer
#*80% (v/v) freshly deionized formamide
#*1X TBE
#**89 mM Trisma base
#**89 mM boric acid
#**2.5 mM Na2EDTA (pH 8.3)
#*10 mM Na2EDTA
#*0.025% (w/v) xylene cyanol
#Heat samples to 100°C for 3 mins
#Load onto prerun gel
#Fractionate by gel electrophoresis on 23% (38:2) polyacrylamide-7M urea gels in salt gradient buffer.
#Continue electrophoresis at constant power of 1.5 W/cm until XC has migrated 17 cm from wells
#Expose and scan in phosphorimager

====Reference====
<biblio>
#Vo-Biochem-2003 pmid=12667071
</biblio>

Talk:Knight:In vitro transcription - Revision history

Reshma P. Shetty at 16:06, 12 December 2006

Reshma P. Shetty: /* Galan et al */

Reshma P. Shetty: /* Vo et al. */

Reshma P. Shetty at 01:45, 7 December 2006