Knight:In vitro transcription protocol

Solutions/reagents: repressorPCR template5X E. coli RNA polymerase transcription bufferTCEP2.5 mM each NTPRNase-free waterE. coli RNA polymerase holoenzymeDNaseI bufferDNase</li></ul> Equipment: Incubator</li>Reaction tubes</li></ul> Steps: <ol>  Prepare template DNA   ''Generate linearized template via PCR. Do a 100 ?L reaction using VF2 and VR. <font color = "#800517">Can be done once, frozen and reused.'' </li> </ol></li>  Option 1: Preincubate repressor and DNA Measure out <font color=#357EC7>20 µl  of <font color=#357EC7>repressor into reaction tube (1). Add <font color=#357EC7>2 µl  of <font color=#357EC7>PCR template. <font color = "#800517">Do the same for relevant controls. </li> Incubate at <font color=#357EC7><b><font color=#357EC7>room temperature  </b> for <font color=#357EC7>2 hrs . </li> Use the following table as a checklist for preparing the reaction in reaction tube (2):  </li>  Incubate at <font color=#357EC7>37°C  for <font color=#357EC7>1 hr . </li> </ol>(or) Option 2: Set up transcription reaction Use the following table as a checklist for preparing the reaction in reaction tube (2): </li>  Incubate at <font color=#357EC7>37°C  for <font color=#357EC7>1 hr . </li> </ol> </li>   DNase treatment (Optional)  <font color = "#800517">This step hasn't been tried. <font color = "#800517">An optional step is to treat the reaction with RNase free DNaseI to remove the template DNA. Measure out <font color=#357EC7>6 µl  of <font color=#357EC7>DNaseI buffer into reaction tube (2). Add <font color=#357EC7>3 µl  of <font color=#357EC7>RNase-free water. Add <font color=#357EC7>1 µl  of <font color=#357EC7>DNase. Incubate at <font color=#357EC7>37°C  for <font color=#357EC7>1 hr . Store at <font color=#357EC7>75°C  for <font color=#357EC7>10 mins . <font color = "#800517">This is to heat-inactivate the enzyme. </li> </ol></ul></ul> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 3 hrs, 10 mins