Knight:In vitro transcription protocol

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<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><p><li><b><font size=3>Prepare template DNA</font></b><br><ol type="a"><p><li><font color = "red"><i>Generate linearized template via PCR. Do a 100 µl reaction using VF2 and VR.</i></font><br><font color = "#800517"><i>Can be done once, frozen and reused.</i></font><br></li></p></ol></li></p><p><li><p><b>Option 1: Preincubate repressor and DNA</b><ol type="a"><p><li>Measure out <b><font color=#357EC7>20 µl</font></b> of <font color=#357EC7>repressor</font> into a reaction tube.<br>Add <b><font color=#357EC7>2 µl</font></b> of <font color=#357EC7>PCR template</font>.<br><font color = "#800517"><i>Do the same for relevant controls.</i></font><br></li></p><p><li>Incubate at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>2 hrs</font></b>.<br></li></p><p><li>Use the following table as a checklist for preparing the reaction:<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>repressor-DNA mixture</font></td><td><font color=#357EC7>5X E. coli RNA polymerase transcription buffer</font></td><td><font color=#357EC7>TCEP</font></td><td><font color=#357EC7>2.5 mM each NTP</font></td><td><font color=#357EC7>E. coli RNA polymerase holoenzyme</font></td><td><font color=#357EC7>RNase-free water</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Reaction</font></td><td><b><b><font color=#357EC7>22 µl</font></b></td><td><b><b><font color=#357EC7>10 µl</font></b></td><td><b><b><font color=#357EC7>0.5 µl</font></b></td><td><b><b><font color=#357EC7>10 µl</font></b></td><td><b><b><font color=#357EC7>2.5 µl</font></b></td><td><b><b><font color=#357EC7>5 µl</font></b></td></tr></body></table></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>1 hr</font></b>.<br></li></p></ol>(or)<p><b>Option 2: Set up transcription reaction</b><ol type="a"><p><li>Use the following table as a checklist for preparing the reaction:<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>RNase-free water</font></td><td><font color=#357EC7>5X E. coli RNA polymerase transcription buffer</font></td><td><font color=#357EC7>TCEP</font></td><td><font color=#357EC7>2.5 mM each NTP</font></td><td><font color=#357EC7>PCR template</font></td><td><font color=#357EC7>E. coli RNA polymerase holoenzyme</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Reaction</font></td><td><b><b><font color=#357EC7>25 µl</font></b></td><td><b><b><font color=#357EC7>10 µl</font></b></td><td><b><b><font color=#357EC7>0.5 µl</font></b></td><td><b><b><font color=#357EC7>10 µl</font></b></td><td><b><b><font color=#357EC7>2 µl</font></b></td><td><b><b><font color=#357EC7>2.5 µl</font></b></td></tr></body></table></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>1 hr</font></b>.<br></li></p></ol></p><p></li></p><p><b><font size=3>DNase treatment (Optional)</font></b><br><font color = "#800517"><i>This step hasn't been tried.</i></font><br><font color = "#800517"><i>An optional step is to treat the reaction with RNase free DNaseI to remove the template DNA.</i></font><br>Add <b><font color=#357EC7>6 µl</font></b> of <font color=#357EC7>DNaseI buffer</font>.<br>Add <b><font color=#357EC7>3 µl</font></b> of <font color=#357EC7>RNase-free water</font>.<br>Add <b><font color=#357EC7>1 µl</font></b> of <font color=#357EC7>DNase</font>.<br>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>1 hr</font></b>.<br>Store at <b><font color=#357EC7>75°C</font></b> for <b><font color=#357EC7>10 mins</font></b>.<br><font color = "#800517"><i>This is to heat-inactivate the enzyme.</i></font><br></li></p></ol>
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<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><p><li><b><font size=3>Prepare template DNA</font></b><br><ol type="a"><p><li><font color = "red"><i>Generate linearized template via PCR. Do a 100 ?L reaction using VF2 and VR.</i></font><br><font color = "#800517"><i>Can be done once, frozen and reused.</i></font><br></li></p></ol></li></p><p><li><p><b>Option 1: Preincubate repressor and DNA</b><ol type="a"><p><li>Measure out <b><font color=#357EC7>20 µl</font></b> of <font color=#357EC7>repressor</font> into reaction tube (1).<br>Add <b><font color=#357EC7>2 µl</font></b> of <font color=#357EC7>PCR template</font>.<br><font color = "#800517"><i>Do the same for relevant controls.</i></font><br></li></p><p><li>Incubate at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>2 hrs</font></b>.<br></li></p><p><li>Use the following table as a checklist for preparing the reaction in reaction tube (2):<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>repressor-DNA mixture</font></td><td><font color=#357EC7>5X E. coli RNA polymerase transcription buffer</font></td><td><font color=#357EC7>TCEP</font></td><td><font color=#357EC7>2.5 mM each NTP</font></td><td><font color=#357EC7>E. coli RNA polymerase holoenzyme</font></td><td><font color=#357EC7>RNase-free water</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Reaction</font></td><td><b><b><font color=#357EC7>22 µl</font></b></td><td><b><b><font color=#357EC7>10 µl</font></b></td><td><b><b><font color=#357EC7>0.5 µl</font></b></td><td><b><b><font color=#357EC7>10 µl</font></b></td><td><b><b><font color=#357EC7>2.5 µl</font></b></td><td><b><b><font color=#357EC7>5 µl</font></b></td></tr></body></table></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>1 hr</font></b>.<br></li></p></ol>(or)<p><b>Option 2: Set up transcription reaction</b><ol type="a"><p><li>Use the following table as a checklist for preparing the reaction in reaction tube (2):<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>RNase-free water</font></td><td><font color=#357EC7>5X E. coli RNA polymerase transcription buffer</font></td><td><font color=#357EC7>TCEP</font></td><td><font color=#357EC7>2.5 mM each NTP</font></td><td><font color=#357EC7>PCR template</font></td><td><font color=#357EC7>E. coli RNA polymerase holoenzyme</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Reaction</font></td><td><b><b><font color=#357EC7>25 µl</font></b></td><td><b><b><font color=#357EC7>10 µl</font></b></td><td><b><b><font color=#357EC7>0.5 µl</font></b></td><td><b><b><font color=#357EC7>10 µl</font></b></td><td><b><b><font color=#357EC7>2 µl</font></b></td><td><b><b><font color=#357EC7>2.5 µl</font></b></td></tr></body></table></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>1 hr</font></b>.<br></li></p></ol></p><p></li></p><p><b><font size=3>DNase treatment (Optional)</font></b><br><font color = "#800517"><i>This step hasn't been tried.</i></font><br><font color = "#800517"><i>An optional step is to treat the reaction with RNase free DNaseI to remove the template DNA.</i></font><br>Measure out <b><font color=#357EC7>6 µl</font></b> of <font color=#357EC7>DNaseI buffer</font> into reaction tube (2).<br>Add <b><font color=#357EC7>3 µl</font></b> of <font color=#357EC7>RNase-free water</font>.<br>Add <b><font color=#357EC7>1 µl</font></b> of <font color=#357EC7>DNase</font>.<br>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>1 hr</font></b>.<br>Store at <b><font color=#357EC7>75°C</font></b> for <b><font color=#357EC7>10 mins</font></b>.<br><font color = "#800517"><i>This is to heat-inactivate the enzyme.</i></font><br></li></p></ol></ul></ul><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 3 hrs, 10 mins</font></b></p>
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Current revision

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 mixture5X E. coli RNA polymerase transcription bufferTCEP2.5 mM each NTPE. coli RNA polymerase holoenzymeRNase-free water
      Reaction22 µl10 µl0.5 µl10 µl2.5 µl5 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 water5X E. coli RNA polymerase transcription bufferTCEP2.5 mM each NTPPCR templateE. coli RNA polymerase holoenzyme
      Reaction25 µl10 µl0.5 µl10 µl2 µl2.5 µl
    2. Incubate at 37°C for 1 hr.

  3. 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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