Knight:In vitro transcription: Difference between revisions
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**10 mM MgCl<sub>2</sub> | **10 mM MgCl<sub>2</sub> | ||
**0.01% Triton® X-100 | **0.01% Triton® X-100 | ||
* | *10 mM DTT | ||
*0. | *0.5 mM NTPs | ||
*1μg T7 D111 DNA as template | *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-<sup>32</sup>P]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 | |||
<biblio> | |||
#Szalewska-Palasz-PNAS-1998 pmid=9539721 | |||
</biblio> |
Revision as of 10:31, 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