Knight:In vitro transcription: Difference between revisions

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==Materials==
==Materials==
*[http://www.epibio.com/item.asp?id=275 E. coli RNA Polymerase Sigma-Saturated Holoenzyme from Epicentre] - ([http://www.epibio.com/pdftechlit/014pl026.pdf protocol])
*[http://www.epibio.com/item.asp?id=275 E. coli RNA Polymerase Sigma-Saturated Holoenzyme from Epicentre] - ([http://www.epibio.com/pdftechlit/014pl026.pdf protocol])
*Linearized template DNA
*NTPs
*DTT


==Reaction conditions==
==Procedure==


===Epicentre===
===Prepare template DNA===
#Generate linearized template via PCR.  Do a 100 μL reaction using VF2 and VR.
#*''Can be done once, frozen and reused.''


====Functional test====
===Option 1: Preincubate repressor and DNA===
50 μL reaction
#Mix
*1X ''E. coli'' RNA polymerase transcription buffer
#*20 μL repressor
**0.04 M Tris-HCl (pH 7.5)
#*2 μL of PCR template
**0.15 M KCl
#**''Do the same for relevant controls.''
**10 mM MgCl<sub>2</sub>
#Incubate 2 hours on benchtop.
**0.01% Triton® X-100
#Make up 50 &mu;L reaction
*2mM DTT
#*22 &mu;L repressor-DNA mixture
*0.25mM NTPs
#*10 &mu;L 5X ''E. coli'' RNA polymerase transcription buffer
*1&mu;g T7 D111 DNA as template
#*0.5 &mu;L of 500 mM TCEP since DTT chelates zinc
#*10 &mu;L of 2.5 mM each NTP
#*5 &mu;L RNase free H<sub>2</sub>O
#*2.5 &mu;L ''E. coli'' RNA polymerase holoenzyme
#Incubate at 37&deg;C for 1 hr.


====Assay test====
===Option 2: Set up transcription reaction===
50 &mu;L reaction
#Make up 50 &mu;L reaction
*1X ''E. coli'' RNA polymerase transcription buffer
#*25 &mu;L RNase free H<sub>2</sub>O
**0.04 M Tris-HCl (pH 7.5)
#*10 &mu;L 5X ''E. coli'' RNA polymerase transcription buffer
**0.15 M KCl
#*0.5 &mu;L of 500 mM TCEP (since DTT chelates zinc)
**10 mM MgCl<sub>2</sub>
#*10 &mu;L of 2.5 mM each NTP
**0.01% Triton® X-100
#*2 &mu;L of PCR template <-perhaps cut this down?  DNA is a pretty bright band?
*10 mM DTT
#*2.5 &mu;L ''E. coli'' RNA polymerase holoenzyme
*0.5 mM NTPs
#Incubate at 37&deg;C for 1 hr.
*1&mu;g T7 D111 DNA as template


===Szalewska-Palasz et al.===
===DNase treatment (optional)===
25 &mu;L reaction
''This step hasn't been tried.''
*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
An optional step is to treat the reaction with [http://www.neb.com/nebecomm/products/productM0303.asp RNase free DNaseI] to remove the template DNA.
#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


====Reference====
#Add 6 &mu;L DNaseI buffer
<biblio>
#Add 3 &mu;L H<sub>2</sub>O
#Szalewska-Palasz-PNAS-1998 pmid=9539721
#Add 1&mu;L DNaseI 
</biblio>
#Incubate 1 hr at 37&deg;C
#Heat inactivate for 10 mins at 75&deg;C
 
====Notes====
*EDTA should be added to a final concentration of 5 mM to protect RNA from being degraded during enzyme inactivation.  But EDTA can chelate magnesium which is needed for DNaseI activity.  So the EDTA may need to be added just prior to inactivation.
*DNase I is not active on DNA bound to proteins.
*DTT which is typically included in ''in vitro'' transcription reactions can chelate zinc.  Therefore, replace DTT with TCEP when the presence of zinc is necessary.


===Galan et al===
===Analyze transcription products===
(Run off transcription assay)
Then analyze via [[Knight:RNA electrophoresis/Native|native agarose gel electrophoresis]].


9 &mu;L reaction
==Controls==
*5nM DNA (supercoiled plasmid)
There are a few controls that can help to ascertain that the assay is working properly.
*100nM CRP
*100nM HpaR or buffer B


*Buffer B
#Template without RNAP
**40 mM Tris-HCl pH 8.0
#*To see what just the DNA template looks like when run on a gel.
**10 mM MgCl<sub>2</sub>
#Template with RNAP
**100 mM KCl
#*To ensure that the linearized template is transcribed by RNAP.
**200 &mu;M cAMP
#Template with RNAP and the buffer that the repressor is resuspended in
**500 &mu;g/mL acetylated BSA
#*To verify that any repression seen is not due to altered salt or pH conditions.


#Incubate reaction at room temperature for 20 mins
==Notes==
#Add 3 &mu;L RNAp at 375 nM in buffer B
#If using either plasmid DNA or DNA template has been linearized by restriction enzyme digestion, Ambion recommends a Proteinase K treatment followed by a phenol:chloroform extraction to eliminate all traces of RNase prior to subsequent reactions. This treatment is necessary because most plasmid DNA has been subjected to RNaseA during purification and restriction enzymes may be contaminated with RNases.
#Incubate 5 mins at 37&deg;C
#However, in the case of PCR generated templates, Ambion adds amplified DNA directly to the transcription reaction with no purification. 5 &mu;l of a 100 &mu;l PCR reaction (or about 0.05 - 0.2 &mu;g of double-stranded DNA) is used as template.  However, with shorter templates or low yields, the concentration of template in a 5 &mu;l aliquot of the crude PCR reaction may be suboptimal. In that case, it may be desirable to concentrate the PCR product by alcohol precipitation.  We do not generally find it necessary to phenol/chloroform extract the PCR reaction before precipitation, although in some cases it may be advisable to do so. <cite>AmbionTemplate</cite>
#Start elongation with 3 &mu;L of prewarmed mixture in buffer B of
#*1mM ATP
#*1mM GTP
#*1mM CTP
#*50&mu;M UTP
#*1&mu;Ci [&alpha;-<sup>32</sup>]UTP
#*500 &mu;g/mL heparin
#Incubate 5 mins at 37&deg;C
#Add 12&mu;L loading buffer containing 1% SDS
#Heat to 70&deg;C
#Electrophorese samples on 7% sequencing gels
#Quantify using phosphorimager


====Reference====
==References==
<biblio>
<biblio>
#Galan-NAR-2003 pmid=14602920
#AmbionTemplate [http://www.ambion.com/techlib/tb/tb_154.html A PCR Strategy for Rapid Generation of Template DNA for Synthesis of Labeled RNA Probes] (contains lots of useful info about generating transcription templates via PCR)
</biblio>
</biblio>
==BioCoder version==
Following is the Knight:In vitro transcription protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder(see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth|Vaishnavi]]
====Text Output====
[[Knight:In vitro transcription protocol]]
====Source Code====
[[Knight:In vitro transcription protocol - source code]]
[[Category:Protocol]]
[[Category:RNA]]
[[Category:In vitro]]

Latest revision as of 02:33, 19 November 2009

Purpose

In vitro transcription using Escherichia coli RNA polymerase.

Materials

Procedure

Prepare template DNA

  1. Generate linearized template via PCR. Do a 100 μL reaction using VF2 and VR.
    • Can be done once, frozen and reused.

Option 1: Preincubate repressor and DNA

  1. Mix
    • 20 μL repressor
    • 2 μL of PCR template
      • Do the same for relevant controls.
  2. Incubate 2 hours on benchtop.
  3. Make up 50 μL reaction
    • 22 μL repressor-DNA mixture
    • 10 μL 5X E. coli RNA polymerase transcription buffer
    • 0.5 μL of 500 mM TCEP since DTT chelates zinc
    • 10 μL of 2.5 mM each NTP
    • 5 μL RNase free H2O
    • 2.5 μL E. coli RNA polymerase holoenzyme
  4. Incubate at 37°C for 1 hr.

Option 2: Set up transcription reaction

  1. Make up 50 μL reaction
    • 25 μL RNase free H2O
    • 10 μL 5X E. coli RNA polymerase transcription buffer
    • 0.5 μL of 500 mM TCEP (since DTT chelates zinc)
    • 10 μL of 2.5 mM each NTP
    • 2 μL of PCR template <-perhaps cut this down? DNA is a pretty bright band?
    • 2.5 μL E. coli RNA polymerase holoenzyme
  2. Incubate at 37°C for 1 hr.

DNase treatment (optional)

This step hasn't been tried.

An optional step is to treat the reaction with RNase free DNaseI to remove the template DNA.

  1. Add 6 μL DNaseI buffer
  2. Add 3 μL H2O
  3. Add 1μL DNaseI
  4. Incubate 1 hr at 37°C
  5. Heat inactivate for 10 mins at 75°C

Notes

  • EDTA should be added to a final concentration of 5 mM to protect RNA from being degraded during enzyme inactivation. But EDTA can chelate magnesium which is needed for DNaseI activity. So the EDTA may need to be added just prior to inactivation.
  • DNase I is not active on DNA bound to proteins.
  • DTT which is typically included in in vitro transcription reactions can chelate zinc. Therefore, replace DTT with TCEP when the presence of zinc is necessary.

Analyze transcription products

Then analyze via native agarose gel electrophoresis.

Controls

There are a few controls that can help to ascertain that the assay is working properly.

  1. Template without RNAP
    • To see what just the DNA template looks like when run on a gel.
  2. Template with RNAP
    • To ensure that the linearized template is transcribed by RNAP.
  3. Template with RNAP and the buffer that the repressor is resuspended in
    • To verify that any repression seen is not due to altered salt or pH conditions.

Notes

  1. If using either plasmid DNA or DNA template has been linearized by restriction enzyme digestion, Ambion recommends a Proteinase K treatment followed by a phenol:chloroform extraction to eliminate all traces of RNase prior to subsequent reactions. This treatment is necessary because most plasmid DNA has been subjected to RNaseA during purification and restriction enzymes may be contaminated with RNases.
  2. However, in the case of PCR generated templates, Ambion adds amplified DNA directly to the transcription reaction with no purification. 5 μl of a 100 μl PCR reaction (or about 0.05 - 0.2 μg of double-stranded DNA) is used as template. However, with shorter templates or low yields, the concentration of template in a 5 μl aliquot of the crude PCR reaction may be suboptimal. In that case, it may be desirable to concentrate the PCR product by alcohol precipitation. We do not generally find it necessary to phenol/chloroform extract the PCR reaction before precipitation, although in some cases it may be advisable to do so. [1]

References

  1. A PCR Strategy for Rapid Generation of Template DNA for Synthesis of Labeled RNA Probes (contains lots of useful info about generating transcription templates via PCR)

    [AmbionTemplate]

BioCoder version

Following is the Knight:In vitro transcription protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder(see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi

Text Output

Knight:In vitro transcription protocol

Source Code

Knight:In vitro transcription protocol - source code

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