Knight:In vitro transcription protocol

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Solutions/reagents:

• repressor
• PCR template
• 5X E. coli RNA polymerase transcription buffer
• TCEP
• 2.5 mM each NTP
• RNase-free water
• E. coli RNA polymerase holoenzyme
• DNaseI buffer
• DNase

Equipment:

• Incubator
• Reaction tubes

Steps:

1. Prepare template DNA
   

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

2. Option 1: Preincubate repressor and DNA

   1. Measure out 20 µl of repressor into reaction tube (1).
      Add 2 µl of PCR template.
      Do the same for relevant controls.
      
   2. Incubate at room temperature for 2 hrs.
      
   3. Use the following table as a checklist for preparing the reaction in reaction tube (2):
      
      

      	repressor-DNA mixture	5X E. coli RNA polymerase transcription buffer	TCEP	2.5 mM each NTP	E. coli RNA polymerase holoenzyme	RNase-free water
      Reaction	22 µl	10 µl	0.5 µl	10 µl	2.5 µl	5 µl

   4. Incubate at 37°C for 1 hr.
      

   (or)

   Option 2: Set up transcription reaction

   1. Use the following table as a checklist for preparing the reaction in reaction tube (2):
      
      

      	RNase-free water	5X E. coli RNA polymerase transcription buffer	TCEP	2.5 mM each NTP	PCR template	E. coli RNA polymerase holoenzyme
      Reaction	25 µl	10 µl	0.5 µl	10 µl	2 µl	2.5 µl

   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.
Measure out 6 µl of DNaseI buffer into reaction tube (2).
Add 3 µl of RNase-free water.
Add 1 µl of DNase.
Incubate at 37°C for 1 hr.
Store at 75°C for 10 mins.
This is to heat-inactivate the enzyme.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 3 hrs, 10 mins