Endy:Colony PCR protocol

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(New page: <html><h2>Solutions/reagents:</h2><ul type="circle"><li>Thermo polymerase buffer</li><li>dNTPS</li><li>Forward primer</li><li>Reverse primer</li><li>Taq or Vent Polymerase</li><li>Template...)
Current revision (02:41, 20 November 2009) (view source)
 
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>Thermo polymerase buffer</li><li>dNTPS</li><li>Forward primer</li><li>Reverse primer</li><li>Taq or Vent Polymerase</li><li>Template suspension</li><li>H<sub>2</sub>O</li></ul><h2>Equipment:</h2><ul type="circle"><li>Thermocycler</li><li>Reaction tubes</li></ul><h2>Steps:</h2><ol><p><li><font color = "#800517"><i>Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 µl sterile water.</i></font><br></li></p><p><li><font color = "#800517"><i>Store the colony resuspension at 4°C so you can start cultures if necessary (should be OK for a couple days, if you need it to last longer you should use an Index plate.)</i></font><br></li></p><p><li><b><font size=3>Reaction mix</font></b><br>Set up a reaction 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>10 µl</font></b> reaction</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Thermo polymerase 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>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>dNTPS</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>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Forward primer</font></td><td><b><font color=#357EC7>40 µM</font></b></td><td><b><font color=#357EC7>0.6 µM</font></b></td><td><b><b><font color=#357EC7>0.15 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Reverse primer</font></td><td><b><font color=#357EC7>40 µM</font></b></td><td><b><font color=#357EC7>0.6 µM</font></b></td><td><b><b><font color=#357EC7>0.15 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Taq or Vent Polymerase</font></td><td><b><font color=#357EC7>--</font></b></td><td><b><font color=#357EC7>--</font></b></td><td><b><b><font color=#357EC7>0.1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Template suspension</font></td><td><b><font color=#357EC7>--</font></b></td><td><b><font color=#357EC7>--</font></b></td><td><b><b><font color=#357EC7>6.6 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>H<sub>2</sub>O</font></td><td><b><font color=#357EC7>--</font></b></td><td><b><font color=#357EC7>--</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></font></b></td></tr></body></table></li></p><p><li><b><font size=3>PCR protocol</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>95°C</font></b>, <b><font color=#357EC7>6 mins</font></b></li></ul>Thermocycling<br><ul><li>No. of cycles: <b><font color=#357EC7>35</font></b></li><li>Denature: <b><font color=#357EC7>95°C</font></b>, <b><font color=#357EC7>30 secs</font></b></li> <li> Anneal: <b><font color=#357EC7>53°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>X</font></b></li></ul>Termination<ul><li>Elongate: <b><font color=#357EC7>72°C</font></b>, <b><font color=#357EC7>10 mins</font></b></li><li>Hold: <b><font color=#357EC7>4°C</font></b>, until removed from machine </li></ul><font color = "#800517"><i>For long amplicons, X = 1 minute + 2.5 s per 100bp.</i></font><br><font color = "#800517"><i>For shorter amplicons, under ~1kb, this can be shortened judiciously.</i></font><br></li></p></ol></html>
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>Thermo polymerase buffer</li><li>dNTPS</li><li>Forward primer</li><li>Reverse primer</li><li>Taq or Vent Polymerase</li><li>Template suspension</li><li>H<sub>2</sub>O</li></ul><h2>Equipment:</h2><ul type="circle"><li>Thermocycler</li><li>Reaction tubes</li></ul><h2>Steps:</h2><ol><p><li><font color = "#800517"><i>Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 ?l sterile water.</i></font><br></li></p><p><li><font color = "#800517"><i>Store the colony resuspension at 4°C so you can start cultures if necessary (should be OK for a couple days, if you need it to last longer you should use an Index plate.</i></font><br></li></p><p><li><b><font size=3>Reaction mix</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>10 µl</font></b> reaction</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Thermo polymerase 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>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>dNTPS</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>1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Forward primer</font></td><td><b><font color=#357EC7>40 µM</font></b></td><td><b><font color=#357EC7>0.6 µM</font></b></td><td><b><b><font color=#357EC7>0.15 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Reverse primer</font></td><td><b><font color=#357EC7>40 µM</font></b></td><td><b><font color=#357EC7>0.6 µM</font></b></td><td><b><b><font color=#357EC7>0.15 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Taq or Vent Polymerase</font></td><td><b><font color=#357EC7>--</font></b></td><td><b><font color=#357EC7>--</font></b></td><td><b><b><font color=#357EC7>0.1 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>Template suspension</font></td><td><b><font color=#357EC7>--</font></b></td><td><b><font color=#357EC7>--</font></b></td><td><b><b><font color=#357EC7>6.6 µl</font></b></font></b></td></tr></body><tbody><tr><td><font color=#357EC7>H<sub>2</sub>O</font></td><td><b><font color=#357EC7>--</font></b></td><td><b><font color=#357EC7>--</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></font></b></td></tr></body></table></li></p><p><li><b><font size=3>PCR protocol</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>95°C</font></b>, <b><font color=#357EC7>6 mins</font></b></li></ul>Thermocycling<br><ul><li>No. of cycles: <b><font color=#357EC7>35</font></b></li><li>Denature: <b><font color=#357EC7>95°C</font></b>, <b><font color=#357EC7>30 secs</font></b></li> <li> Anneal: <b><font color=#357EC7>53°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>60 secs</font></b></li></ul>Termination<ul><li>Elongate: <b><font color=#357EC7>72°C</font></b>, <b><font color=#357EC7>10 mins</font></b></li><li>Hold: <b><font color=#357EC7>4°C</font></b>, until removed from machine </li></ul><font color = "#800517"><i>For long amplicons, elongation time = 1 minute + 2.5 s per 100bp.</i></font><br><font color = "#800517"><i>For shorter amplicons, under ~1kb, this can be shortened judiciously.</i></font><br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 1 hr, 28 mins</font></b></p></html>

Current revision

Solutions/reagents:

  • Thermo polymerase buffer
  • dNTPS
  • Forward primer
  • Reverse primer
  • Taq or Vent Polymerase
  • Template suspension
  • H2O

Equipment:

  • Thermocycler
  • Reaction tubes

Steps:

  1. Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 ?l sterile water.
  2. Store the colony resuspension at 4°C so you can start cultures if necessary (should be OK for a couple days, if you need it to last longer you should use an Index plate.
  3. Reaction mix
    Set up a reaction in reaction tube (1) as follows on ice:

     Initial concentrationFinal concentrationFinal volume per 10 µl reaction
    Thermo polymerase buffer10X1X1 µl
    dNTPS10X1X1 µl
    Forward primer40 µM0.6 µM0.15 µl
    Reverse primer40 µM0.6 µM0.15 µl
    Taq or Vent Polymerase----0.1 µl
    Template suspension----6.6 µl
    H2O----1 µl
  4. PCR protocol
    Program a standard thermocycler to run the reaction using the following parameters:
    Initial denaturation
    • Denature: 95°C, 6 mins
    Thermocycling
    • No. of cycles: 35
    • Denature: 95°C, 30 secs
    • Anneal: 53°C, 30 secs
    • Elongate: 72°C, 60 secs
    Termination
    • Elongate: 72°C, 10 mins
    • Hold: 4°C, until removed from machine
    For long amplicons, elongation time = 1 minute + 2.5 s per 100bp.
    For shorter amplicons, under ~1kb, this can be shortened judiciously.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 28 mins

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