Team:Alberta/Project/ByteAmplification

From 2009.igem.org

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<h2>Byte PCR and Digestion:</h2>
<h2>Byte PCR and Digestion:</h2>
 +
 +
1) In an PCR Tube mix the following (Assuming a 100 μL Reaction Volume):
<ul>
<ul>
-
     <li>Pipet 150 uL 50% glycerol into 3* properly labeled 1.5 ml screw cap tubes.
+
     <li>4 μL Template (pAB or pBA – 1.5 ng/μL)
-
     <li>Add 350 uL of overnight culture to each tube.
+
     <li>4 μL Forward Universal Primer (10 μM)
-
     <li>Pipet up and down to gently mix.
+
     <li>4 μL Reverse Universal Primer (10 μM)
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     <li>Flash freeze in liquid N2 or dry ice/methanol bath
+
     <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>

Revision as of 03:44, 20 October 2009

University of Alberta - BioBytes










































































































Byte Amplification and End Preparation

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:

  • Overnight with bacterial growth
  • Screw cap tubes
  • Glycerol

Byte PCR and Digestion:

1) In an 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

Ethanol Precipitation

  • If we make extra we have them if we need to send them out or want to store in several locations.

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