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</div>
 
  
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<div style = "padding: 35px;">
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<p style= "font-size: 50px;"> BioSensor Lab </p>
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 +
<h3>A1: T7 salt sensor </h3>
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<h4> Introduction </h4>
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<p> The goal is amplify T7 salt sensor, digest it along with pSB1C3 plasmid with EcoRI and PstI</p>
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<h4>Materials </h4>
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<ul>
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<li> Polymerase: Fusion Taq or Ampli tas (gold) </li>
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<li>Primers: PrefixF  and SuffixR  </li>
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<li>Nuclease free H2O </li>
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<li>5xHF PCR Buffer </li>
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<li>gBlock DNA </li>
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<li> dNTPs</li>
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<li>PCR tubes </li>
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</ul>
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<h4> Procedure </h4>
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<p style= "font-size: 20px;> Preparation of reagents </p>
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<ol>
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<li> Resuspend gBLOCK in 20µL nuclease free dH2O</li>
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<li> Resuspend primers to make 100µM stock then dilute to make a 10µM stock</li>
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</ol>
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<p style= "font-size: 20px;> PCR </p>
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<ol>
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<li>Add the following reagents to a PCR tube: </li>
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<li>Run the PCR machine </li>
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</ol>
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<p style= "font-size: 20px;> Digestion and Cloning </p>
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<ol>
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<li> Digest amplified sequence with EcoRI and PstI </li>
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<li> Digest pSB1C3 vector with EcoRI and PstI</li>
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<li> If you clone into a vector that expresses a fluorescent protein, other than red since you are cloning in red, then colonies that still express that fluorescent protein will be negative clones and the red or white colonies will be the ones that you want.</li>
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<li>Run on an agarose gel, cut out the bands and purify.  </li>
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<li> Send clones for sequencing  </li>
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</ol>
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</div>
  
  

Revision as of 21:20, 23 October 2017

BioSensor Lab

A1: T7 salt sensor

Introduction

The goal is amplify T7 salt sensor, digest it along with pSB1C3 plasmid with EcoRI and PstI

Materials

  • Polymerase: Fusion Taq or Ampli tas (gold)
  • Primers: PrefixF and SuffixR
  • Nuclease free H2O
  • 5xHF PCR Buffer
  • gBlock DNA
  • dNTPs
  • PCR tubes

Procedure

PCR

  1. Add the following reagents to a PCR tube:
  2. Run the PCR machine

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