Talk:Knight:In vitro transcription

Epicentre
From Epicentre.

Functional test
50 &mu;L 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
 * 1&mu;g template

Assay test
50 &mu;L 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
 * 1&mu;g template

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

Procedure

 * 1) Add repressor
 * 2) Incubate 5min at 37&deg;C
 * 3) Transfer samples to ice bath
 * 4) Add 1 unit E. coli RNAP from Epicentre
 * 5) Add 150 &mu;M CTP and GTP
 * 6) Add 1 mM ATP
 * 7) Add 15 &mu;M UTP
 * 8) Add [&alpha;-32P]UTP to 1mCi/mL
 * 9) Incubate samples at 37&deg;C for 12.5 mins
 * 10) Stop reaction with equal volume of
 * 11) *BSA (1.2mg/mL)
 * 12) *0.1 mM EDTA, pH 8.0
 * 13) *5.1 M ammonium acetate
 * 14) Transfer to ice bath
 * 15) Precipitate RNA with 2 volumes 96% ethanol in liquid nitrogen
 * 16) Centrifuge in microcentrifuge at maximum speed for 30 mins
 * 17) Dry pellet
 * 18) Resuspend in 20 &mu;L of
 * 19) *98% formamide
 * 20) *0.25% bromophenol blue
 * 21) *0.25% xylene cyanol
 * 22) Incubate at 65&deg;C for 5 mins
 * 23) Electrophorese in 6% polyacrylamide gel containing 46% urea in TBE buffer at 30mA
 * 24) Dry the gel
 * 25) Visualize RNA bands by autoradiography and quantify via densitometry

Reference

 * 1) Szalewska-Palasz-PNAS-1998 pmid=9539721

Galan et al.
(Run off transcription assay)

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 MgCl2
 * 100 mM KCl
 * 200 &mu;M cAMP
 * 500 &mu;g/mL acetylated BSA

Procedure

 * 1) Incubate reaction at room temperature for 20 mins
 * 2) Add 3 &mu;L RNAp at 375 nM in buffer B
 * 3) Incubate 5 mins at 37&deg;C
 * 4) Start elongation with 3 &mu;L of prewarmed mixture in buffer B of
 * 5) *1mM ATP
 * 6) *1mM GTP
 * 7) *1mM CTP
 * 8) *50&mu;M UTP
 * 9) *1&mu;Ci [&alpha;-32]UTP
 * 10) *500 &mu;g/mL heparin
 * 11) Incubate 5 mins at 37&deg;C
 * 12) Add 12&mu;L loading buffer containing 1% SDS
 * 13) Heat to 70&deg;C
 * 14) Electrophorese samples on 7% sequencing gels
 * 15) Quantify using phosphorimager

Reference

 * 1) Galan-NAR-2003 pmid=14602920

Marschall et al.
Run off transcription assay


 * 1) Used either supercoiled or linear template
 * 2) 5 &mu;L of 20nM DNA template in potassium glutamate solution
 * 3) *40 mM Hepes (pH 8.0)
 * 4) *10 mM magnesium chloride
 * 5) *100 mM potassium glutamate
 * 6) *200 &mu;M cAMP
 * 7) *500 &mu;g/mL acetylated bovine serum albumin
 * 8) Add 5 &mu;L of 400 nM Lrp or buffer
 * 9) Add 5 &mu;L of 400 nM Crp or buffer
 * 10) Incubate at room temperature for 15 mins
 * 11) 7&mu;L of mixture was incubated at 37&deg;C for 5 mins
 * 12) Add 3.5 &mu;L RNAp at 260 nM
 * 13) Incubate for 5 mins at 37&deg;C
 * 14) Start polymerization by adding 3.5 &mu;L prewarmed mixture containing
 * 15) *1mM ATP
 * 16) *1mM GTP
 * 17) *1mM CTP
 * 18) *50&mu;M UTP
 * 19) *500 &mu;g/mL heparin
 * 20) *1&mu;Ci [&alpha;-32]UTP
 * 21) Incubate 5 mins (what temp?)
 * 22) Stop reaction by adding 20mM EDTA in formamide containing xylene cyanol and bromophenol blue
 * 23) Heat to 65&deg;C
 * 24) Electrophorese samples on 7% sequencing gels
 * 25) Autoradiograph and quantify using phosphorimager

Reference

 * 1) Marschall-JMB-1998 pmid=9512707

Vo et al.
Steady state transcription assay

20-50&mu;L reaction mixtures (prepped on ice)
 * 30 nM promoter template
 * 150 mM KCl
 * 100&mu;M each of four NTPs
 * [&gamma;-32P]ATP (~5cpm/fmol) in buffer
 * 40 mM Tris-HCl pH8
 * 10 mM MgCl2
 * 10 mM &beta;-mercaptoethanol
 * 10 &mu;g/mL acetylated BSA
 * 1) Add E. coli RNA polymerase to final concentration of 30 nM
 * 2) Incubate at 37&deg;C
 * 3) Remove 5-10 &mu;L samples at 0-60 mins
 * 4) Mix with equal volume of formamide loading buffer
 * 5) *80% (v/v) freshly deionized formamide
 * 6) *1X TBE
 * 7) **89 mM Trisma base
 * 8) **89 mM boric acid
 * 9) **2.5 mM Na2EDTA (pH 8.3)
 * 10) *10 mM Na2EDTA
 * 11) *0.025% (w/v) xylene cyanol
 * 12) Heat samples to 100&deg;C for 3 mins
 * 13) Load onto prerun gel
 * 14) Fractionate by gel electrophoresis on 23% (38:2) polyacrylamide-7M urea gels in salt gradient buffer.
 * 15) Continue electrophoresis at constant power of 1.5 W/cm until XC has migrated 17 cm from wells
 * 16) Expose and scan in phosphorimager

Reference

 * 1) Vo-Biochem-2003 pmid=12667071