Team:BYU Provo/Notebook/CommonProcedures

From 2014.igem.org

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<h2>TAQ PCR (50 uL Reaction)</h2>
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<blockquote><h2>TAQ PCR (50 uL Reaction)</h2>
<ul><li>35ul ddH20</li>
<ul><li>35ul ddH20</li>
<li>10ul 5X REDTAQ buffer (mix well before use!!)</li>
<li>10ul 5X REDTAQ buffer (mix well before use!!)</li>
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<li>Repeat (2-4) 35 times</li>
<li>Repeat (2-4) 35 times</li>
<li>72°C for 5 min</li>
<li>72°C for 5 min</li>
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<li>4°C for forever</li></ol>
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<li>4°C for forever</li></ol></blockquote>
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<h2>Q5 PCR Reaction (50 uL Reaction)</h2>
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<blockquote><h2>Q5 PCR Reaction (50 uL Reaction)</h2>
<ul><li>10uL 5x Q5 Reaction Buffer</li>
<ul><li>10uL 5x Q5 Reaction Buffer</li>
<li>1uL dNTPs</li>
<li>1uL dNTPs</li>
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<li>1-2uL Template</li>
<li>1-2uL Template</li>
<li>Add 0.5 uL Q5 Polymerase right before you start the PCR reaction.</li></ul>
<li>Add 0.5 uL Q5 Polymerase right before you start the PCR reaction.</li></ul>
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<p>Set up reactions on ice, making sure to keep Q5 Polymerase on ice until needed.</p>
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<p>Set up reactions on ice, making sure to keep Q5 Polymerase on ice until needed.</p></blockquote>
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<h2>Phusion PCR</h2>
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<blockquote><h2>Phusion PCR</h2>
<ul><li>35ul ddH20</li>
<ul><li>35ul ddH20</li>
<li>10ul 5X Phusion GC buffer</li>
<li>10ul 5X Phusion GC buffer</li>
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<li>Repeat (2-4) 35 times</li>
<li>Repeat (2-4) 35 times</li>
<li>72°C for 5 min</li>
<li>72°C for 5 min</li>
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<li>4°C for forever</li></ol>
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<li>4°C for forever</li></ol></blockquote>
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<h2>Standard 1% Agarose Gel</h2>
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<blockquote><h2>Standard 1% Agarose Gel</h2>
<ul><li>75ml of 1X TAE buffer and 0.75 grams of agarose (regular agarose, not low melt).  Warm in microwave for about 60-90 seconds or until the agarose is completely dissolved.</li>
<ul><li>75ml of 1X TAE buffer and 0.75 grams of agarose (regular agarose, not low melt).  Warm in microwave for about 60-90 seconds or until the agarose is completely dissolved.</li>
<li>With gloves on, add 1 drop (~6ul of 1mg/ml) ethidium bromide and swirl to mix.</li>
<li>With gloves on, add 1 drop (~6ul of 1mg/ml) ethidium bromide and swirl to mix.</li>
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<li>Allow the flask to cool until the glass feels warm/hot, then pour into gel bed. Insert appropriate comb and allow to cool until solid.</li></ul>
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<li>Allow the flask to cool until the glass feels warm/hot, then pour into gel bed. Insert appropriate comb and allow to cool until solid.</li></ul></blockquote>
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<h2>Low-melt Agarose Gel</h2>
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<blockquote><h2>Low-melt Agarose Gel</h2>
<ul><li>Mix 0.75g of low-melt agarose with 75mL of TAE. Caution: Low-melt heats more quickly than standard agarose in the microwave.</li>
<ul><li>Mix 0.75g of low-melt agarose with 75mL of TAE. Caution: Low-melt heats more quickly than standard agarose in the microwave.</li>
<li>Add 5ul of ethidium bromide to the agarose solution before casting</li>
<li>Add 5ul of ethidium bromide to the agarose solution before casting</li>
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<li>Use a large-tooth comb to form wells that will accommodate ~40-50µl of material.</li></ul>
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<li>Use a large-tooth comb to form wells that will accommodate ~40-50µl of material.</li></ul></blockquote>
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<h2>Analysis of PCR Products by Agarose Electrophoresis</h2>
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<blockquote><h2>Analysis of PCR Products by Agarose Electrophoresis</h2>
<ul><li>Add 6uL of 10X loading dye (orange dye) to each 50ul PCR product.</li>
<ul><li>Add 6uL of 10X loading dye (orange dye) to each 50ul PCR product.</li>
<li>Move the gel into the proper orientation in the gel box. Cover your gel with 1X TAE buffer. Add 6uL of DNA reference ladder to the first well.</li>
<li>Move the gel into the proper orientation in the gel box. Cover your gel with 1X TAE buffer. Add 6uL of DNA reference ladder to the first well.</li>
<li>Add 6uL of each PCR product into subsequent wells.</li>
<li>Add 6uL of each PCR product into subsequent wells.</li>
<li>Turn on the gel box power supply and run at 150-175 volts.  It will take about 15-30 minutes to complete depending on the desired resolution.</li>
<li>Turn on the gel box power supply and run at 150-175 volts.  It will take about 15-30 minutes to complete depending on the desired resolution.</li>
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<li>Gels can be visualized and recorded in the Alpha-Imager.</li>
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<li>Gels can be visualized and recorded in the Alpha-Imager.</li></blockquote>
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Revision as of 04:32, 22 July 2014


BYU 2014 Notebook

EDIT Procedures

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TAQ PCR (50 uL Reaction)

  • 35ul ddH20
  • 10ul 5X REDTAQ buffer (mix well before use!!)
  • 1.0ul 10mM dNTP’s
  • 1ul each Primer (50 uM stock)
  • 1ul appropriate diluted template DNA
  • Mix well then add 2.5ul REDTAQ polymerase

Set up reactions on ice, and keep them on ice until placing them on the PCR machine which has been pre-warmed to 94°C. Abundant templates only require 20-25 cycles for amplification; dilute/complex templates require 35-40 cycles. Extension times vary depending on target size.

  • Standard PCR program:
  1. 95°C for 2 min
  2. 95°C for 30 sec
  3. 55°C for 30 sec
  4. 72°C for 2 min
  5. Repeat (2-4) 35 times
  6. 72°C for 5 min
  7. 4°C for forever

Q5 PCR Reaction (50 uL Reaction)

  • 10uL 5x Q5 Reaction Buffer
  • 1uL dNTPs
  • 1uL Forward Primer
  • 1uL Reverse Primer
  • 10uL Q5 Enhancer
  • 23.5uL ddH2O
  • 1-2uL Template
  • Add 0.5 uL Q5 Polymerase right before you start the PCR reaction.

Set up reactions on ice, making sure to keep Q5 Polymerase on ice until needed.

Phusion PCR

  • 35ul ddH20
  • 10ul 5X Phusion GC buffer
  • 1.0uL DMSO
  • 1.5ul 10 mM dNTP’s
  • 1ul each Primer (50 uM stock)
  • 1ul appropriate diluted template DNA
  • 0.5ul Phusion Polymerase
  • Standard Phusion PCR program:
  1. 95°C for 2 min
  2. 95°C for 30 sec
  3. 55°C for 30 sec
  4. 72°C for 2 min
  5. Repeat (2-4) 35 times
  6. 72°C for 5 min
  7. 4°C for forever

Standard 1% Agarose Gel

  • 75ml of 1X TAE buffer and 0.75 grams of agarose (regular agarose, not low melt). Warm in microwave for about 60-90 seconds or until the agarose is completely dissolved.
  • With gloves on, add 1 drop (~6ul of 1mg/ml) ethidium bromide and swirl to mix.
  • Allow the flask to cool until the glass feels warm/hot, then pour into gel bed. Insert appropriate comb and allow to cool until solid.

Low-melt Agarose Gel

  • Mix 0.75g of low-melt agarose with 75mL of TAE. Caution: Low-melt heats more quickly than standard agarose in the microwave.
  • Add 5ul of ethidium bromide to the agarose solution before casting
  • Use a large-tooth comb to form wells that will accommodate ~40-50µl of material.

Analysis of PCR Products by Agarose Electrophoresis

  • Add 6uL of 10X loading dye (orange dye) to each 50ul PCR product.
  • Move the gel into the proper orientation in the gel box. Cover your gel with 1X TAE buffer. Add 6uL of DNA reference ladder to the first well.
  • Add 6uL of each PCR product into subsequent wells.
  • Turn on the gel box power supply and run at 150-175 volts. It will take about 15-30 minutes to complete depending on the desired resolution.
  • Gels can be visualized and recorded in the Alpha-Imager.




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