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:
- Prepare template DNA
- Generate linearized template via PCR. Do a 100 ?L reaction using VF2 and VR.
Can be done once, frozen and reused.
- Generate linearized template via PCR. Do a 100 ?L reaction using VF2 and VR.
Option 1: Preincubate repressor and DNA
- Measure out 20 µl of repressor into a reaction tube.
Add 2 µl of PCR template.
Do the same for relevant controls. - Incubate at room temperature for 2 hrs.
- Use the following table as a checklist for preparing the reaction:
<thead></thead><tbody></body>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 - Incubate at 37°C for 1 hr.
Option 2: Set up transcription reaction
- Use the following table as a checklist for preparing the reaction:
<thead></thead><tbody></body>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 - Incubate at 37°C for 1 hr.
- Measure out 20 µl of repressor into a reaction tube.
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.
Add 6 µl of DNaseI buffer.
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.
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