Knight:In vitro transcription

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==Materials==
==Materials==
*[http://www.epibio.com/item.asp?id=275 E. coli RNA Polymerase Sigma-Saturated Holoenzyme from Epicentre] - ([http://www.epibio.com/pdftechlit/014pl026.pdf protocol])
*[http://www.epibio.com/item.asp?id=275 E. coli RNA Polymerase Sigma-Saturated Holoenzyme from Epicentre] - ([http://www.epibio.com/pdftechlit/014pl026.pdf protocol])
 +
*Linearized template DNA
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==Reaction conditions==
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==Procedure==
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===Epicentre===
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===Prepare template DNA===
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#Generate linearized template via either PCR or restriction digest. 
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#Ambion recommends a Proteinase K treatment followed by a phenol:chloroform extraction to eliminate all traces of RNase prior to subsequent reactions. (Note that if the DNA template has been linearized by restriction enzyme digestion, a similar treatment is recommended, since restriction enzymes may be contaminated with RNases.)
 +
 
 +
 
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===Set up reaction===
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From Epicentre
====Functional test====
====Functional test====
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*2mM DTT
*2mM DTT
*0.25mM NTPs
*0.25mM NTPs
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*1μg T7 D111 DNA as template
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*1μg template
====Assay test====
====Assay test====
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*10 mM DTT
*10 mM DTT
*0.5 mM NTPs
*0.5 mM NTPs
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*1μg T7 D111 DNA as template
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*1μg template
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===Szalewska-Palasz et al.===
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====25 μL reaction====
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*Buffer M
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**20 mM Hepes, pH 8.0
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**5 mM magnesium acetate
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**4 mM DTT
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**1 mM EDTA
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**1mM ATP
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**BSA (5mg/mL)
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**0.2% Triton X-100
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**5% glycerol
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*5 μg template DNA
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====Procedure====
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#Add repressor
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#Incubate 5min at 37°C
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-
#Transfer samples to ice bath
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#Add 1 unit ''E. coli'' RNAP from Epicentre
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-
#Add 150 μM CTP and GTP
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-
#Add 1 mM ATP
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#Add 15 μM UTP
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#Add [&alpha;-<sup>32</sup>P]UTP to 1mCi/mL
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#Incubate samples at 37&deg;C for 12.5 mins
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#Stop reaction with equal volume of
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#*BSA (1.2mg/mL)
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#*0.1 mM EDTA, pH 8.0
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-
#*5.1 M ammonium acetate
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#Transfer to ice bath
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#Precipitate RNA with 2 volumes 96% ethanol in liquid nitrogen
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#Centrifuge in microcentrifuge at maximum speed for 30 mins
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#Dry pellet
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#Resuspend in 20 &mu;L of
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#*98% formamide
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#*0.25% bromophenol blue
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#*0.25% xylene cyanol
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#Incubate at 65&deg;C for 5 mins
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#Electrophorese in 6% polyacrylamide gel containing 46% urea in TBE buffer at 30mA
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#Dry the gel
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#Visualize RNA bands by autoradiography and quantify via densitometry
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====Reference====
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<biblio>
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#Szalewska-Palasz-PNAS-1998 pmid=9539721
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</biblio>
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===Galan et al===
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(Run off transcription assay)
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====9 &mu;L reaction====
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*5nM DNA (supercoiled plasmid)
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*100nM CRP
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*100nM HpaR or buffer B
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*Buffer B
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**40 mM Tris-HCl pH 8.0
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**10 mM MgCl<sub>2</sub>
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**100 mM KCl
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**200 &mu;M cAMP
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**500 &mu;g/mL acetylated BSA
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-
 
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====Procedure====
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#Incubate reaction at room temperature for 20 mins
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#Add 3 &mu;L RNAp at 375 nM in buffer B
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#Incubate 5 mins at 37&deg;C
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#Start elongation with 3 &mu;L of prewarmed mixture in buffer B of
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#*1mM ATP
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#*1mM GTP
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#*1mM CTP
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#*50&mu;M UTP
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#*1&mu;Ci [&alpha;-<sup>32</sup>]UTP
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#*500 &mu;g/mL heparin
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#Incubate 5 mins at 37&deg;C
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#Add 12&mu;L loading buffer containing 1% SDS
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#Heat to 70&deg;C
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#Electrophorese samples on 7% sequencing gels
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#Quantify using phosphorimager
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====Reference====
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<biblio>
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#Galan-NAR-2003 pmid=14602920
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</biblio>
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===Marschall et al.===
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Run off transcription assay
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#Used either supercoiled or linear template
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#5 &mu;L of 20nM DNA template in potassium glutamate solution
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#*40 mM Hepes (pH 8.0)
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#*10 mM magnesium chloride
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#*100 mM potassium glutamate
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#*200 &mu;M cAMP
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#*500 &mu;g/mL acetylated bovine serum albumin
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#Add 5 &mu;L of 400 nM Lrp or buffer
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#Add 5 &mu;L of 400 nM Crp or buffer
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#Incubate at room temperature for 15 mins
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#7&mu;L of mixture was incubated at 37&deg;C for 5 mins
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#Add 3.5 &mu;L RNAp at 260 nM
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#Incubate for 5 mins at 37&deg;C
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#Start polymerization by adding 3.5 &mu;L prewarmed mixture containing
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#*1mM ATP
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#*1mM GTP
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#*1mM CTP
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#*50&mu;M UTP
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#*500 &mu;g/mL heparin
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#*1&mu;Ci [&alpha;-<sup>32</sup>]UTP
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#Incubate 5 mins (what temp?)
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#Stop reaction by adding 20mM EDTA in formamide containing xylene cyanol and bromophenol blue
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#Heat to 65&deg;C
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#Electrophorese samples on 7% sequencing gels
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#Autoradiograph and quantify using phosphorimager
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====Reference====
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<biblio>
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#Marschall-JMB-1998 pmid=9512707
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</biblio>
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===Vo et al.===
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Steady state transcription assay
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20-50&mu;L reaction mixtures (prepped on ice)
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*30 nM promoter template
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*150 mM KCl
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*100&mu;M each of four NTPs
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*[&gamma;-<sup32</sup>P]ATP (~5cpm/fmol) in buffer
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**40 mM Tris-HCl pH8
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**10 mM MgCl<sub>2</sub>
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**10 mM &beta;-mercaptoethanol
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**10 &mu;g/mL acetylated BSA
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#Add E. coli RNA polymerase to final concentration of 30 nM
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#Incubate at 37&deg;C
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#Remove 5-10 &mu;L samples at 0-60 mins
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#Mix with equal volume of formamide loading buffer
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#*80% (v/v) freshly deionized formamide
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#*1X TBE
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#**89 mM Trisma base
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#**89 mM boric acid
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#**2.5 mM Na<sub>2</sub>EDTA (pH 8.3)
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#*10 mM Na<sub>2</sub>EDTA
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#*0.025% (w/v) xylene cyanol
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#Heat samples to 100&deg;C for 3 mins
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#Load onto prerun gel
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#Fractionate by gel electrophoresis on 23% (38:2) polyacrylamide-7M urea gels in salt gradient buffer.
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#Continue electrophoresis at constant power of 1.5 W/cm until XC has migrated 17 cm from wells
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#Expose and scan in phosphorimager
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====Reference====
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Then analyze via [[Knight:RNA electrophoresis/Native|native agarose gel electrophoresis]].
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<biblio>
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#Vo-Biochem-2003 pmid=12667071
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-
</biblio>
+

Revision as of 20:50, 6 December 2006

Contents

Purpose

In vitro transcription using Escherichia coli RNA polymerase.

Materials

Procedure

Prepare template DNA

  1. Generate linearized template via either PCR or restriction digest.
  2. Ambion recommends a Proteinase K treatment followed by a phenol:chloroform extraction to eliminate all traces of RNase prior to subsequent reactions. (Note that if the DNA template has been linearized by restriction enzyme digestion, a similar treatment is recommended, since restriction enzymes may be contaminated with RNases.)


Set up reaction

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 MgCl2
    • 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 MgCl2
    • 0.01% Triton® X-100
  • 10 mM DTT
  • 0.5 mM NTPs
  • 1μg template

Then analyze via native agarose gel electrophoresis.

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