Knight:In vitro transcription

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 27: Line 27:
**10 mM MgCl<sub>2</sub>
**10 mM MgCl<sub>2</sub>
**0.01% Triton® X-100
**0.01% Triton® X-100
-
*10mM DTT
+
*10 mM DTT
-
*0.5mM NTPs
+
*0.5 mM NTPs
*1&mu;g T7 D111 DNA as template
*1&mu;g T7 D111 DNA as template
 +
 +
===Szalewska-Palasz et al.===
 +
25 &mu;L 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 &mu;g template DNA
 +
 +
 +
#Add repressor
 +
#Incubate 5min at 37&deg;C
 +
#Transfer samples to ice bath
 +
#Add 1 unit ''E. coli'' RNAP from Epicentre
 +
#Add 150 &mu;M CTP and GTP
 +
#Add 1 mM ATP
 +
#Add 15 &mu;M UTP
 +
#Add [&alpha;-<sup>32</sup>P]UTP to 1mCi/mL
 +
#Incubate samples at 37&deg;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 &mu;L of
 +
#*98% formamide
 +
#*0.25% bromophenol blue
 +
#*0.25% xylene cyanol
 +
#Incubate at 65&deg;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
 +
<biblio>
 +
#Szalewska-Palasz-PNAS-1998 pmid=9539721
 +
</biblio>

Revision as of 12:31, 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]
Personal tools