Talk:Knight:In vitro transcription
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==Other reaction conditions from the literature== | ==Other reaction conditions from the literature== | ||
| + | |||
| + | ===Epicentre=== | ||
| + | From 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 MgCl<sub>2</sub> | ||
| + | **0.01% Triton® X-100 | ||
| + | *2mM DTT | ||
| + | *0.25mM NTPs | ||
| + | *1μg 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 MgCl<sub>2</sub> | ||
| + | **0.01% Triton® X-100 | ||
| + | *10 mM DTT | ||
| + | *0.5 mM NTPs | ||
| + | *1μg template | ||
| + | |||
===Szalewska-Palasz et al.=== | ===Szalewska-Palasz et al.=== | ||
| Line 46: | Line 72: | ||
</biblio> | </biblio> | ||
| - | ===Galan et al=== | + | ===Galan et al.=== |
(Run off transcription assay) | (Run off transcription assay) | ||
| Line 124: | Line 150: | ||
*150 mM KCl | *150 mM KCl | ||
*100μM each of four NTPs | *100μM each of four NTPs | ||
| - | *[γ-< | + | *[γ-<sup>32</sup>P]ATP (~5cpm/fmol) in buffer |
**40 mM Tris-HCl pH8 | **40 mM Tris-HCl pH8 | ||
**10 mM MgCl<sub>2</sub> | **10 mM MgCl<sub>2</sub> | ||
Current revision
Contents |
Other reaction conditions from the literature
Epicentre
From 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 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 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
- 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
Reference
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
- Incubate reaction at room temperature for 20 mins
- Add 3 μL RNAp at 375 nM in buffer B
- Incubate 5 mins at 37°C
- 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
- Incubate 5 mins at 37°C
- Add 12μL loading buffer containing 1% SDS
- Heat to 70°C
- Electrophorese samples on 7% sequencing gels
- Quantify using phosphorimager
Reference
- Galán B, Kolb A, Sanz JM, García JL, and Prieto MA. . pmid:14602920.
Marschall et al.
Run off transcription assay
- Used either supercoiled or linear template
- 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
- Add 5 μL of 400 nM Lrp or buffer
- Add 5 μL of 400 nM Crp or buffer
- Incubate at room temperature for 15 mins
- 7μL of mixture was incubated at 37°C for 5 mins
- Add 3.5 μL RNAp at 260 nM
- Incubate for 5 mins at 37°C
- 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
- Incubate 5 mins (what temp?)
- Stop reaction by adding 20mM EDTA in formamide containing xylene cyanol and bromophenol blue
- Heat to 65°C
- Electrophorese samples on 7% sequencing gels
- Autoradiograph and quantify using phosphorimager
Reference
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-32P]ATP (~5cpm/fmol) in buffer
- 40 mM Tris-HCl pH8
- 10 mM MgCl2
- 10 mM β-mercaptoethanol
- 10 μg/mL acetylated BSA
- Add E. coli RNA polymerase to final concentration of 30 nM
- Incubate at 37°C
- Remove 5-10 μL 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 Na2EDTA (pH 8.3)
- 10 mM Na2EDTA
- 0.025% (w/v) xylene cyanol
- Heat samples to 100°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
- Vo NV, Hsu LM, Kane CM, and Chamberlin MJ. . pmid:12667071.
