Polymerase Chain Reaction (PCR) protocol

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(New page: <html> <h2>Solutions/reagents:</h2><ul type="circle"><li>Buffer</li><li>dNTPs</li><li>MgCl <sub>2</sub></li><li>DMSO</li><li>Upstream primer</li><li>Downstream primer</li><li>Template DNA<...)
Current revision (02:54, 20 November 2009) (view source)
 
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<h2>Solutions/reagents:</h2><ul type="circle"><li>Buffer</li><li>dNTPs</li><li>MgCl <sub>2</sub></li><li>DMSO</li><li>Upstream primer</li><li>Downstream primer</li><li>Template DNA</li><li>dH<sub>2</sub>O</li><li>DNA Polymerase</li></ul><h2>Equipment:</h2><ul type="circle"><li>Thermocycler</li><li>Reaction tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>For Promega GoTaq:</font></b><br>Set up a reaction in reaction tube (1) as follows on ice:<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>Initial concentration</font></td><td><font color=#357EC7>Final concentration</font></td><td><font color=#357EC7>Final volume per <b><font color=#357EC7>50 µl</font></b> reaction</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Buffer</font></td><td><b><font color=#357EC7>5X</font></b></td><td><b><font color=#357EC7>1X</font></b></td><td><b><b><font color=#357EC7>10 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>dNTPs</font></td><td><b><font color=#357EC7>1.25mM</font></b></td><td><b><font color=#357EC7>0.25mM</font></b></td><td><b><b><font color=#357EC7>10 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>MgCl <sub>2</sub></font></td><td><b><font color=#357EC7>25mM</font></b></td><td><b><font color=#357EC7>2.5mM</font></b></td><td><b><b><font color=#357EC7>5 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>DMSO</font></td><td><b><font color=#357EC7>50%</font></b></td><td><b><font color=#357EC7>5%</font></b></td><td><b><b><font color=#357EC7>5 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Upstream primer</font></td><td><b><font color=#357EC7>50pmol</font></b></td><td><b><font color=#357EC7>1pmol</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Downstream primer</font></td><td><b><font color=#357EC7>50pmol</font></b></td><td><b><font color=#357EC7>1pmol</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Template DNA</font></td><td><b><font color=#357EC7>??</font></b></td><td><b><font color=#357EC7>~0.5µg</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>dH<sub>2</sub>O</font></td><td><b><font color=#357EC7>N/A</font></b></td><td><b><font color=#357EC7>N/A</font></b></td><td><b><b><font color=#357EC7>16.75 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>DNA Polymerase</font></td><td><b><font color=#357EC7>5u/µL</font></b></td><td><b><font color=#357EC7>1.25u</font></b></td><td><b><b><font color=#357EC7>0.25 µl</font></b></font></b></td></tr></body></table></li></p><p><li><b><font size=3>For Roche High Fidelity:</font></b><br>Set up a reaction in reaction tube (1) as follows on ice:<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>Initial concentration</font></td><td><font color=#357EC7>Final concentration</font></td><td><font color=#357EC7>Final volume per <b><font color=#357EC7>50 µl</font></b> reaction</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Buffer</font></td><td><b><font color=#357EC7>10X</font></b></td><td><b><font color=#357EC7>1X</font></b></td><td><b><b><font color=#357EC7>5 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>dNTPs</font></td><td><b><font color=#357EC7>1.25mM</font></b></td><td><b><font color=#357EC7>0.25mM</font></b></td><td><b><b><font color=#357EC7>10 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>DMSO</font></td><td><b><font color=#357EC7>50%</font></b></td><td><b><font color=#357EC7>5%</font></b></td><td><b><b><font color=#357EC7>5 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Upstream primer</font></td><td><b><font color=#357EC7>50pmol</font></b></td><td><b><font color=#357EC7>1pmol</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Downstream primer</font></td><td><b><font color=#357EC7>50pmol</font></b></td><td><b><font color=#357EC7>1pmol</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Template DNA</font></td><td><b><font color=#357EC7>??</font></b></td><td><b><font color=#357EC7>~0.5µg</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>dH<sub>2</sub>O</font></td><td><b><font color=#357EC7>N/A</font></b></td><td><b><font color=#357EC7>N/A</font></b></td><td><b><b><font color=#357EC7>16.75 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>DNA Polymerase</font></td><td><b><font color=#357EC7>5u/µL</font></b></td><td><b><font color=#357EC7>2.5u</font></b></td><td><b><b><font color=#357EC7>0.5 µl</font></b></font></b></td></tr></body></table></li></p><p><li><b><font size=3>An example program:</font></b><br>Program a standard thermocycler to run the reaction using the following parameters:<br>Initial denaturation<br><ul><li>Denature: <b><font color=#357EC7>96°C</font></b>, <b><font color=#357EC7>5 mins</font></b></li></ul><font color = "#800517"><i>This denatures any double stranded DNA.</i></font><br>Thermocycling<br><ul><li>No. of cycles: <b><font color=#357EC7>25</font></b></li><li>Denature: <b><font color=#357EC7>96°C</font></b>, <b><font color=#357EC7>1 min</font></b></li> <li> Anneal: <b><font color=#357EC7>55°C</font></b>, <b><font color=#357EC7>30 secs</font></b></li> <li>Elongate: <b><font color=#357EC7>72°C</font></b>, <b><font color=#357EC7>1 min</font></b></li></ul>Termination<ul><li>Elongate: <b><font color=#357EC7>72°C</font></b>, <b><font color=#357EC7>5 mins</font></b></li><li>Hold: <b><font color=#357EC7>4°C</font></b>, until removed from machine </li></ul><font color = "#800517"><i>Final extension for incomplete strands and holding of sample at low temperature.</i></font><br></li></p></ol>
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<h2>Solutions/reagents:</h2><ul type="circle"><li>Buffer</li><li>dNTPs</li><li>MgCl <sub>2</sub></li><li>DMSO</li><li>Upstream primer</li><li>Downstream primer</li><li>Template DNA</li><li>dH<sub>2</sub>O</li><li>DNA Polymerase</li></ul><h2>Equipment:</h2><ul type="circle"><li>Thermocycler</li><li>Reaction tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>For Promega GoTaq:</font></b><br>Set up a reaction in reaction tube (1) as follows on ice:<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>Initial concentration</font></td><td><font color=#357EC7>Final concentration</font></td><td><font color=#357EC7>Final volume per <b><font color=#357EC7>50 µl</font></b> reaction</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Buffer</font></td><td><b><font color=#357EC7>5X</font></b></td><td><b><font color=#357EC7>1X</font></b></td><td><b><b><font color=#357EC7>10 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>dNTPs</font></td><td><b><font color=#357EC7>1.25mM</font></b></td><td><b><font color=#357EC7>0.25mM</font></b></td><td><b><b><font color=#357EC7>10 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>MgCl <sub>2</sub></font></td><td><b><font color=#357EC7>25mM</font></b></td><td><b><font color=#357EC7>2.5mM</font></b></td><td><b><b><font color=#357EC7>5 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>DMSO</font></td><td><b><font color=#357EC7>50%</font></b></td><td><b><font color=#357EC7>5%</font></b></td><td><b><b><font color=#357EC7>5 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Upstream primer</font></td><td><b><font color=#357EC7>50pmol</font></b></td><td><b><font color=#357EC7>1pmol</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Downstream primer</font></td><td><b><font color=#357EC7>50pmol</font></b></td><td><b><font color=#357EC7>1pmol</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Template DNA</font></td><td><b><font color=#357EC7>??</font></b></td><td><b><font color=#357EC7>~0.5µg</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>dH<sub>2</sub>O</font></td><td><b><font color=#357EC7>N/A</font></b></td><td><b><font color=#357EC7>N/A</font></b></td><td><b><b><font color=#357EC7>16.75 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>DNA Polymerase</font></td><td><b><font color=#357EC7>5u/?L</font></b></td><td><b><font color=#357EC7>1.25u</font></b></td><td><b><b><font color=#357EC7>0.25 µl</font></b></font></b></td></tr></body></table></li></p><p><li><b><font size=3>For Roche High Fidelity:</font></b><br>Set up a reaction in reaction tube (1) as follows on ice:<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>Initial concentration</font></td><td><font color=#357EC7>Final concentration</font></td><td><font color=#357EC7>Final volume per <b><font color=#357EC7>50 µl</font></b> reaction</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Buffer</font></td><td><b><font color=#357EC7>10X</font></b></td><td><b><font color=#357EC7>1X</font></b></td><td><b><b><font color=#357EC7>5 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>dNTPs</font></td><td><b><font color=#357EC7>1.25mM</font></b></td><td><b><font color=#357EC7>0.25mM</font></b></td><td><b><b><font color=#357EC7>10 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>DMSO</font></td><td><b><font color=#357EC7>50%</font></b></td><td><b><font color=#357EC7>5%</font></b></td><td><b><b><font color=#357EC7>5 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Upstream primer</font></td><td><b><font color=#357EC7>50pmol</font></b></td><td><b><font color=#357EC7>1pmol</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Downstream primer</font></td><td><b><font color=#357EC7>50pmol</font></b></td><td><b><font color=#357EC7>1pmol</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Template DNA</font></td><td><b><font color=#357EC7>??</font></b></td><td><b><font color=#357EC7>~0.5µg</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>dH<sub>2</sub>O</font></td><td><b><font color=#357EC7>N/A</font></b></td><td><b><font color=#357EC7>N/A</font></b></td><td><b><b><font color=#357EC7>16.75 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>DNA Polymerase</font></td><td><b><font color=#357EC7>5u/?L</font></b></td><td><b><font color=#357EC7>2.5u</font></b></td><td><b><b><font color=#357EC7>0.5 µl</font></b></font></b></td></tr></body></table></li></p><p><li><b><font size=3>An example program:</font></b><br>Program a standard thermocycler to run the reaction using the following parameters:<br>Initial denaturation<br><ul><li>Denature: <b><font color=#357EC7>96°C</font></b>, <b><font color=#357EC7>5 mins</font></b></li></ul><font color = "#800517"><i>This denatures any double stranded DNA.</i></font><br>Thermocycling<br><ul><li>No. of cycles: <b><font color=#357EC7>25</font></b></li><li>Denature: <b><font color=#357EC7>96°C</font></b>, <b><font color=#357EC7>1 min</font></b></li> <li> Anneal: <b><font color=#357EC7>55°C</font></b>, <b><font color=#357EC7>30 secs</font></b></li> <li>Elongate: <b><font color=#357EC7>72°C</font></b>, <b><font color=#357EC7>1 min</font></b></li></ul>Termination<ul><li>Elongate: <b><font color=#357EC7>72°C</font></b>, <b><font color=#357EC7>5 mins</font></b></li><li>Hold: <b><font color=#357EC7>4°C</font></b>, until removed from machine </li></ul><font color = "#800517"><i>Final extension for incomplete strands and holding of sample at low temperature.</i></font><br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 1 hr, 15 mins</font></b></p>
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Current revision

Solutions/reagents:

  • Buffer
  • dNTPs
  • MgCl 2
  • DMSO
  • Upstream primer
  • Downstream primer
  • Template DNA
  • dH2O
  • DNA Polymerase

Equipment:

  • Thermocycler
  • Reaction tubes

Steps:

  1. For Promega GoTaq:
    Set up a reaction in reaction tube (1) as follows on ice:

     Initial concentrationFinal concentrationFinal volume per 50 µl reaction
    Buffer5X1X10 µl
    dNTPs1.25mM0.25mM10 µl
    MgCl 225mM2.5mM5 µl
    DMSO50%5%5 µl
    Upstream primer50pmol1pmol1 µl
    Downstream primer50pmol1pmol1 µl
    Template DNA??~0.5µg1 µl
    dH2ON/AN/A16.75 µl
    DNA Polymerase5u/?L1.25u0.25 µl
  2. For Roche High Fidelity:
    Set up a reaction in reaction tube (1) as follows on ice:

     Initial concentrationFinal concentrationFinal volume per 50 µl reaction
    Buffer10X1X5 µl
    dNTPs1.25mM0.25mM10 µl
    DMSO50%5%5 µl
    Upstream primer50pmol1pmol1 µl
    Downstream primer50pmol1pmol1 µl
    Template DNA??~0.5µg1 µl
    dH2ON/AN/A16.75 µl
    DNA Polymerase5u/?L2.5u0.5 µl
  3. An example program:
    Program a standard thermocycler to run the reaction using the following parameters:
    Initial denaturation
    • Denature: 96°C, 5 mins
    This denatures any double stranded DNA.
    Thermocycling
    • No. of cycles: 25
    • Denature: 96°C, 1 min
    • Anneal: 55°C, 30 secs
    • Elongate: 72°C, 1 min
    Termination
    • Elongate: 72°C, 5 mins
    • Hold: 4°C, until removed from machine
    Final extension for incomplete strands and holding of sample at low temperature.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 15 mins

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