Knight:In vitro transcription: Difference between revisions
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**5% glycerol | **5% glycerol | ||
*5 μg template DNA | *5 μg template DNA | ||
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#Visualize RNA bands by autoradiography and quantify via densitometry | #Visualize RNA bands by autoradiography and quantify via densitometry | ||
====Reference==== | |||
<biblio> | <biblio> | ||
#Szalewska-Palasz-PNAS-1998 pmid=9539721 | #Szalewska-Palasz-PNAS-1998 pmid=9539721 | ||
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===Galan et al=== | ===Galan et al=== | ||
(Run off transcription assay) | |||
9 μL reaction | 9 μL reaction | ||
*5nM DNA (supercoiled plasmid) | *5nM DNA (supercoiled plasmid) | ||
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#Quantify using phosphorimager | #Quantify using phosphorimager | ||
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<biblio> | <biblio> | ||
#Galan-NAR-2003 pmid=14602920 | #Galan-NAR-2003 pmid=14602920 | ||
</biblio> | </biblio> |
Revision as of 10:44, 28 November 2006
Purpose
In vitro transcription using Escherichia coli RNA polymerase.
Materials
Reaction conditions
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 MgCl2
- 0.01% Triton® X-100
- 2mM DTT
- 0.25mM NTPs
- 1μg T7 D111 DNA as 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 MgCl2
- 0.01% Triton® X-100
- 10 mM DTT
- 0.5 mM NTPs
- 1μg T7 D111 DNA as template
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
- 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
- Szalewska-Pałasz A, Wegrzyn A, Błaszczak A, Taylor K, and Wegrzyn G. DnaA-stimulated transcriptional activation of orilambda: Escherichia coli RNA polymerase beta subunit as a transcriptional activator contact site. Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4241-6. DOI:10.1073/pnas.95.8.4241 |
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
- 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