Knight:In vitro transcription

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<biblio>
<biblio>
#Szalewska-Palasz-PNAS-1998 pmid=9539721
#Szalewska-Palasz-PNAS-1998 pmid=9539721
 +
</biblio>
 +
 +
===Galan et al===
 +
9 &mu;L reaction
 +
*5nM DNA (supercoiled plasmid)
 +
*100nM CRP
 +
*100nM HpaR or buffer B
 +
 +
*Buffer B
 +
**40 mM Tris-HCl pH 8.0
 +
**10 mM MgCl<sub>2</sub>
 +
**100 mM KCl
 +
**200 &mu;M cAMP
 +
**500 &mu;g/mL acetylated BSA
 +
 +
#Incubate reaction at room temperature for 20 mins
 +
#Add 3 &mu;L RNAp at 375 nM in buffer B
 +
#Incubate 5 mins at 37&deg;C
 +
#Start elongation with 3 &mu;L of prewarmed mixture in buffer B of
 +
#*1mM ATP
 +
#*1mM GTP
 +
#*1mM CTP
 +
#*50&mu;M UTP
 +
#*1&mu;Ci [&alpha;-<sup>32</sup>]UTP
 +
#*500 &mu;g/mL heparin
 +
#Incubate 5 mins at 37&deg;C
 +
#Add 12&mu;L loading buffer containing 1% SDS
 +
#Heat to 70&deg;C
 +
#Electrophorese samples on 7% sequencing gels
 +
#Quantify using phosphorimager
 +
 +
<biblio>
 +
#Galan-NAR-2003 pmid=14602920
</biblio>
</biblio>

Revision as of 12:42, 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


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

Galan et al

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