Knight:In vitro transcription
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(Difference between revisions)
| 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 | ||
| - | * | + | *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 13:31, 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
- 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
