Knight:In vitro transcription

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(Marschall et al.)
Line 145: Line 145:
<biblio>
<biblio>
#Marschall-JMB-1998 pmid=9512707
#Marschall-JMB-1998 pmid=9512707
 +
</biblio>
 +
 +
===Vo et al.===
 +
Steady state transcription assay
 +
 +
20-50&mu;L reaction mixtures (prepped on ice)
 +
*30 nM promoter template
 +
*150 mM KCl
 +
*100&mu;M each of four NTPs
 +
*[&gamma;-<sup32</sup>P]ATP (~5cpm/fmol) in buffer
 +
**40 mM Tris-HCl pH8
 +
**10 mM MgCl<sub>2</sub>
 +
**10 mM &beta;-mercaptoethanol
 +
**10 &mu;g/mL acetylated BSA
 +
#Add E. coli RNA polymerase to final concentration of 30 nM
 +
#Incubate at 37&deg;C
 +
#Remove 5-10 &mu;L 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 Na<sub>2</sub>EDTA (pH 8.3)
 +
#*10 mM Na<sub>2</sub>EDTA
 +
#*0.025% (w/v) xylene cyanol
 +
#Heat samples to 100&deg;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>
</biblio>

Revision as of 13:11, 28 November 2006

Contents

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

Procedure

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

Reference

  1. Szalewska-Pałasz A, Wegrzyn A, Błaszczak A, Taylor K, and Wegrzyn G. . pmid:9539721. PubMed HubMed [Szalewska-Palasz-PNAS-1998]

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

  1. Incubate reaction at room temperature for 20 mins
  2. Add 3 μL RNAp at 375 nM in buffer B
  3. Incubate 5 mins at 37°C
  4. 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
  5. Incubate 5 mins at 37°C
  6. Add 12μL loading buffer containing 1% SDS
  7. Heat to 70°C
  8. Electrophorese samples on 7% sequencing gels
  9. Quantify using phosphorimager

Reference

  1. Galán B, Kolb A, Sanz JM, García JL, and Prieto MA. . pmid:14602920. PubMed HubMed [Galan-NAR-2003]

Marschall et al.

Run off transcription assay

  1. Used either supercoiled or linear template
  2. 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
  3. Add 5 μL of 400 nM Lrp or buffer
  4. Add 5 μL of 400 nM Crp or buffer
  5. Incubate at room temperature for 15 mins
  6. 7μL of mixture was incubated at 37°C for 5 mins
  7. Add 3.5 μL RNAp at 260 nM
  8. Incubate for 5 mins at 37°C
  9. 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
  10. Incubate 5 mins (what temp?)
  11. Stop reaction by adding 20mM EDTA in formamide containing xylene cyanol and bromophenol blue
  12. Heat to 65°C
  13. Electrophorese samples on 7% sequencing gels
  14. Autoradiograph and quantify using phosphorimager

Reference

  1. Marschall C, Labrousse V, Kreimer M, Weichart D, Kolb A, and Hengge-Aronis R. . pmid:9512707. PubMed HubMed [Marschall-JMB-1998]

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-<sup32</sup>P]ATP (~5cpm/fmol) in buffer
    • 40 mM Tris-HCl pH8
    • 10 mM MgCl2
    • 10 mM β-mercaptoethanol
    • 10 μg/mL acetylated BSA
  1. Add E. coli RNA polymerase to final concentration of 30 nM
  2. Incubate at 37°C
  3. Remove 5-10 μL samples at 0-60 mins
  4. 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
  5. Heat samples to 100°C for 3 mins
  6. Load onto prerun gel
  7. Fractionate by gel electrophoresis on 23% (38:2) polyacrylamide-7M urea gels in salt gradient buffer.
  8. Continue electrophoresis at constant power of 1.5 W/cm until XC has migrated 17 cm from wells
  9. Expose and scan in phosphorimager

Reference

  1. Vo NV, Hsu LM, Kane CM, and Chamberlin MJ. . pmid:12667071. PubMed HubMed [Vo-Biochem-2003]
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