Knight:In vitro transcription protocol

<html> <h2>Solutions/reagents:</h2><ul type="circle"><li>repressor</li><li>PCR template</li><li>5X E. coli RNA polymerase transcription buffer</li><li>TCEP</li><li>2.5 mM each NTP</li><li>RNase-free water</li><li>E. coli RNA polymerase holoenzyme</li><li>DNaseI buffer</li><li>DNase</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Reaction tubes</li></ul><h2>Steps:</h2><ol><li>Prepare template DNA <ol type="a"><li>Generate linearized template via PCR. Do a 100 ?L reaction using VF2 and VR. Can be done once, frozen and reused. </li></ol></li><li>Option 1: Preincubate repressor and DNA<ol type="a"><li>Measure out 20 µl of repressor into reaction tube (1). Add 2 µl of PCR template. Do the same for relevant controls. </li><li>Incubate at room temperature for 2 hrs. </li><li>Use the following table as a checklist for preparing the reaction in reaction tube (2): <table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td> </td><td>repressor-DNA mixture</td><td>5X E. coli RNA polymerase transcription buffer</td><td>TCEP</td><td>2.5 mM each NTP</td><td>E. coli RNA polymerase holoenzyme</td><td>RNase-free water</td></tr></thead><tbody><tr><td>Reaction</td><td>22 µl</td><td>10 µl</td><td>0.5 µl</td><td>10 µl</td><td>2.5 µl</td><td>5 µl</td></tr></body></table></li><li>Incubate at 37°C for 1 hr. </li></ol>(or)Option 2: Set up transcription reaction<ol type="a"><li>Use the following table as a checklist for preparing the reaction in reaction tube (2): <table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td> </td><td>RNase-free water</td><td>5X E. coli RNA polymerase transcription buffer</td><td>TCEP</td><td>2.5 mM each NTP</td><td>PCR template</td><td>E. coli RNA polymerase holoenzyme</td></tr></thead><tbody><tr><td>Reaction</td><td>25 µl</td><td>10 µl</td><td>0.5 µl</td><td>10 µl</td><td>2 µl</td><td>2.5 µl</td></tr></body></table></li><li>Incubate at 37°C for 1 hr. </li></ol></li>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. </li></ol></ul></ul>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 3 hrs, 10 mins </html>

Knight:In vitro transcription protocol

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