Difference between revisions of "Team:Northwestern/ligation"

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     <ul>
 
     <ul>
 
       <li>Phosphorylate, ligate and transform immediately after each other<br></li>
 
       <li>Phosphorylate, ligate and transform immediately after each other<br></li>
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      <li>Modified Phosphorylation Procedure:</li>
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    <ul> <li>Modified Phosphorylation Procedure:</li>
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       <ul><ul>
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       <ul>
 
         <li>150 ng DNA</li>
 
         <li>150 ng DNA</li>
 
         <li>3 uL T4 ligase buffer</li>
 
         <li>3 uL T4 ligase buffer</li>

Latest revision as of 20:05, 16 October 2016

Protocols

Phosphorylation and Ligation

  • Phosphorylate, ligate and transform immediately after each other
    • Modified Phosphorylation Procedure:
      • 150 ng DNA
      • 3 uL T4 ligase buffer
      • 1 uL T4 PNK
      • Water to 30 uL
    • Run at 37°C for 1 hour
  • Ligation: (into storage vectors-pSB1K3)
    • 20 uL reaction volume:
      • 50 ng backbone (linearized pSB1K3 at 25 ng/uL from iGEM)
      • *Use NEBioCalculator, 3:1 insert:backbone molar ratio
      • 2 uL T4 ligase buffer
      • 1 uL T4 ligase
      • Water to 20 uL
    • Run for 15 minutes (max 30) at room temperature
    • Heat to inactivate at 80°C for 20 minutes or transform immediately