Team:Alberta/Project/ByteAmplification

From 2009.igem.org

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     <h1>Byte Amplification and End Preparation</h1>
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     <h1>Byte Production via PCR and USER Digestion</h1>
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<P>
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This procedure allows for the amplification of any gene or part in the pAB and pBA plasmids.  The procedure is the same for both pAB and pBA Byte Amplifcation with the only difference being the universal primers (for pAB Bytes use pAB+ and pAB-, for pBA Bytes use pBA+ and pBA-).
 +
</P>
<h2>What you will need:</h2>
<h2>What you will need:</h2>
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<ul>
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<ul><li>PFU Turbo Cx (Stratagene)
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<li>Overnight with bacterial growth
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     <li>FWD/REV Primers
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     <li>Screw cap tubes
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     <li>DNA Template
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     <li>Glycerol
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<li>dNTPs
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<li>ddH2O
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<li>USER™
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<li>Gel Purification Kit
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<li>EtOH (100%, 70%)
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<li>NaOAc 3M pH 5.2
</ul>
</ul>
<h2>Byte PCR and Digestion:</h2>
<h2>Byte PCR and Digestion:</h2>
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1. In a PCR tube mix the following (Assuming a 100 μL Reaction Volume):
<ul>
<ul>
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     <li>Pipet 150 uL 50% glycerol into 3* properly labeled 1.5 ml screw cap tubes.
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     <li>4 μL Template (pAB or pBA – 1.5 ng/μL)
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     <li>Add 350 uL of overnight culture to each tube.
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     <li>4 μL Forward Universal Primer (10 μM)
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     <li>Pipet up and down to gently mix.
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     <li>4 μL Reverse Universal Primer (10 μM)
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     <li>Flash freeze in liquid N2 or dry ice/methanol bath
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     <li>2 μL PFU Cx (2.5 U/μL)
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     <li>Place in -80 C freezer when frozen.
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     <li>10 μL PFU Cx Buffer (10X)
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    <li>10 μL dNTPs (2mM)
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    <li>66 μL ddH2O
</ul>
</ul>
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2. Run the PCR reaction under the following conditions:
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<ul>
 +
<li>95˚C (2 Minutes)
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        <li>30 Cycles of:
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<ol><li>95˚C (30 Seconds)</li>
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<li>56˚C (1 Minute)</li>
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<li>72˚C (2 Minutes)</li></ol>
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        <li>72˚C (5 Minutes)</li>
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 +
</ul>
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 +
3. Add 1 μL USER™ (New England Biolabs) to each PCR tube and incubate at 37˚C for 1 hour.
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<BR>4. Run the PCR product on an agarose gel.
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<BR>5. Gel column purify to isolate PCR product (add the 10 μL 3M sodium acetate prior to loading onto column)
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<BR>6.      (ethanol precipitation may be required).
 +
<h2>Ethanol Precipitation</h2>
<h2>Ethanol Precipitation</h2>
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<ul><li> If we make extra we have them if we need to send them out or want to store in several locations.</ul>
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 +
1. In a 1.5 mL microcentrifuge tube add 1/10th volume of 3M sodium acetate, pH 5.2 to DNA solution to be precipitated.
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<BR>2. Add 2x volume 100% ethanol to DNA + sodium acetate solution.
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<BR>3. Incubate at 0 or -20˚C for at least 30 minutes (the longer, the better).
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<BR>4. Centrifuge at 4˚C for 30 min at 14000 rpm.
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<BR>5. Aspirate supernatant carefully.
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<BR>6. Add 750 L 70% ethanol to tube, vortex briefly to wash pellet.
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<BR>7. Centrifuge at 14000 rpm for at least 10 minutes at 4˚C.
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<BR>8. Aspirate supernatant carefully.
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<BR>9. Resuspend pellet in minimal volume of ddH2O or 10 mM Tris-HCl pH 8.5 buffer.
 +
 

Latest revision as of 20:19, 21 October 2009

University of Alberta - BioBytes










































































































Byte Production via PCR and USER Digestion

This procedure allows for the amplification of any gene or part in the pAB and pBA plasmids. The procedure is the same for both pAB and pBA Byte Amplifcation with the only difference being the universal primers (for pAB Bytes use pAB+ and pAB-, for pBA Bytes use pBA+ and pBA-).

What you will need:

  • PFU Turbo Cx (Stratagene)
  • FWD/REV Primers
  • DNA Template
  • dNTPs
  • ddH2O
  • USER™
  • Gel Purification Kit
  • EtOH (100%, 70%)
  • NaOAc 3M pH 5.2

Byte PCR and Digestion:

1. In a PCR tube mix the following (Assuming a 100 μL Reaction Volume):
  • 4 μL Template (pAB or pBA – 1.5 ng/μL)
  • 4 μL Forward Universal Primer (10 μM)
  • 4 μL Reverse Universal Primer (10 μM)
  • 2 μL PFU Cx (2.5 U/μL)
  • 10 μL PFU Cx Buffer (10X)
  • 10 μL dNTPs (2mM)
  • 66 μL ddH2O
2. Run the PCR reaction under the following conditions:
  • 95˚C (2 Minutes)
  • 30 Cycles of:
    1. 95˚C (30 Seconds)
    2. 56˚C (1 Minute)
    3. 72˚C (2 Minutes)
  • 72˚C (5 Minutes)
3. Add 1 μL USER™ (New England Biolabs) to each PCR tube and incubate at 37˚C for 1 hour.
4. Run the PCR product on an agarose gel.
5. Gel column purify to isolate PCR product (add the 10 μL 3M sodium acetate prior to loading onto column)
6. (ethanol precipitation may be required).

Ethanol Precipitation

1. In a 1.5 mL microcentrifuge tube add 1/10th volume of 3M sodium acetate, pH 5.2 to DNA solution to be precipitated.
2. Add 2x volume 100% ethanol to DNA + sodium acetate solution.
3. Incubate at 0 or -20˚C for at least 30 minutes (the longer, the better).
4. Centrifuge at 4˚C for 30 min at 14000 rpm.
5. Aspirate supernatant carefully.
6. Add 750 L 70% ethanol to tube, vortex briefly to wash pellet.
7. Centrifuge at 14000 rpm for at least 10 minutes at 4˚C.
8. Aspirate supernatant carefully.
9. Resuspend pellet in minimal volume of ddH2O or 10 mM Tris-HCl pH 8.5 buffer.

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