Team:BYU Provo/Notebook/CommonProcedures

From 2014.igem.org

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<li>Gels can be visualized and recorded in the Alpha-Imager.</li></ul>
<li>Gels can be visualized and recorded in the Alpha-Imager.</li></ul>
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<h2>Restriction Digest of Plasmid</h2>
+
<h2>Restriction Digest of Plasmid Insert</h2>
<ul><li>PCR product (insert) digestion (50 uL reaction)</li>
<ul><li>PCR product (insert) digestion (50 uL reaction)</li>
<li>6 µl 10X Cutsmart NEB buffer</li>
<li>6 µl 10X Cutsmart NEB buffer</li>
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<p>(**always mix reagents well before adding enzyme as the final reagent)</p>
<p>(**always mix reagents well before adding enzyme as the final reagent)</p>
<ul><li>1.5 µl of each restriction enzyme (PstI/HindIII)</li></ul>
<ul><li>1.5 µl of each restriction enzyme (PstI/HindIII)</li></ul>
 +
<p>After all of these are mixed together they must be placed in the 37ºC bath or incubator for at least 1 hour.</p>
 +
 +
<h2>Restriction Digest of Plasmid Vector</h2>
 +
<ul><li>~14 µl H2O</li>
 +
<li>5 µl Cutsmart NEB buffer</li>
 +
<li>20 µl DNA vector (pBAD, pLAT plasmid)</li></ul>
 +
<p>(**always mix reagents well before adding enzyme as the final reagent)</p>
 +
<ul><li>1.5 µl of each restriction enzyme  (PstI/HindIII)</li></ul>
 +
<p>After all of these are mixed together they must be placed in the 37ºC bath or incubator for at least 1 hour.</p>
 +
 +
 +
<h2>Nano-Drop</h2>
<h2>Nano-Drop</h2>

Revision as of 21:45, 30 July 2014


BYU 2014 Notebook

EDIT Procedures

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RedTAQ 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

TAQ Polymerase PCR (25 uL Reaction)

  • 2.5 uL Thermopol buffer
  • 1 uL dNTPs
  • 1 uL Forward Primer
  • 1 uL Reverse Primer
  • 18.5 uL ddH2O
  • 1-2uL Template
  • Add 0.25 uL TAQ Polymerase at the end right before you start the PCR reaction.

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.

Restriction Digest of Plasmid Insert

  • PCR product (insert) digestion (50 uL reaction)
  • 6 µl 10X Cutsmart NEB buffer
  • 50 µl (all of ) PCR product

(**always mix reagents well before adding enzyme as the final reagent)

  • 1.5 µl of each restriction enzyme (PstI/HindIII)

After all of these are mixed together they must be placed in the 37ºC bath or incubator for at least 1 hour.

Restriction Digest of Plasmid Vector

  • ~14 µl H2O
  • 5 µl Cutsmart NEB buffer
  • 20 µl DNA vector (pBAD, pLAT plasmid)

(**always mix reagents well before adding enzyme as the final reagent)

  • 1.5 µl of each restriction enzyme (PstI/HindIII)

After all of these are mixed together they must be placed in the 37ºC bath or incubator for at least 1 hour.

Nano-Drop

    Performing a nano-drop on your purified plasmid allows you to determine the concentration and purity of your plasmid

  • Clean lens with kimiwipe
  • Place 2 uL ddH2O on the lens, follow instructions on the program
  • Clean lens with wipe and place 2 uL of calibration buffer on the lens. Click the "blank" option.
  • Clean lens with wipe and place 2 uL of sample, then click "measure" in the program.