Knight:In vitro transcription

Purpose
In vitro transcription using Escherichia coli RNA polymerase.

Materials

 * E. coli RNA Polymerase Sigma-Saturated Holoenzyme from Epicentre - (protocol)
 * Linearized template DNA
 * NTPs
 * DTT

Prepare template DNA

 * 1) Generate linearized template via PCR. Do a 100 &mu;L reaction using VF2 and VR.
 * 2) *Can be done once, frozen and reused.

Option 1: Preincubate repressor and DNA

 * Mix
 * 1) *20 &mu;L repressor
 * 2) *2 &mu;L of PCR template
 * 3) **Do the same for relevant controls.
 * 4) Incubate 2 hours on benchtop.
 * 5) Make up 50 &mu;L reaction
 * 6) *22 &mu;L repressor-DNA mixture
 * 7) *10 &mu;L 5X E. coli RNA polymerase transcription buffer
 * 8) *0.5 &mu;L of 500 mM TCEP since DTT chelates zinc
 * 9) *10 &mu;L of 2.5 mM each NTP
 * 10) *5 &mu;L RNase free H2O
 * 11) *2.5 &mu;L E. coli RNA polymerase holoenzyme
 * 12) Incubate at 37&deg;C for 1 hr.

Option 2: Set up transcription reaction

 * 1) Make up 50 &mu;L reaction
 * 2) *25 &mu;L RNase free H2O
 * 3) *10 &mu;L 5X E. coli RNA polymerase transcription buffer
 * 4) *0.5 &mu;L of 500 mM TCEP (since DTT chelates zinc)
 * 5) *10 &mu;L of 2.5 mM each NTP
 * 6) *2 &mu;L of PCR template <-perhaps cut this down? DNA is a pretty bright band?
 * 7) *2.5 &mu;L E. coli RNA polymerase holoenzyme
 * 8) Incubate at 37&deg;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 &mu;L DNaseI buffer
 * 2) Add 3 &mu;L H2O
 * 3) Add 1&mu;L DNaseI
 * 4) Incubate 1 hr at 37&deg;C
 * 5) Heat inactivate for 10 mins at 75&deg;C

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
 * 2) *To see what just the DNA template looks like when run on a gel.
 * 3) Template with RNAP
 * 4) *To ensure that the linearized template is transcribed by RNAP.
 * 5) Template with RNAP and the buffer that the repressor is resuspended in
 * 6) *To verify that any repression seen is not due to altered salt or pH conditions.

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