Knight:In vitro transcription protocol: Difference between revisions
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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 | <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> </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> </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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Latest revision as of 06:20, 20 November 2009
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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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