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 reaction tube (1).
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 in reaction tube (2):
<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 in reaction tube (2):
<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 reaction tube (1).
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
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