Engineering BioBrick vectors from BioBrick parts/DNA ligation

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(New page: ==Materials== *[http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA ligase] from [http://www.neb.com/ NEB] *Deionized, sterile H<sub>2</sub>O *Purified, linearized destination ve...)
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==Materials==
==Materials==
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*[http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA ligase] from [http://www.neb.com/ NEB]
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*[http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA Ligase] from [http://www.neb.com/ NEB]
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*10X T4 DNA Ligase buffer
*Deionized, sterile H<sub>2</sub>O
*Deionized, sterile H<sub>2</sub>O
*Purified, linearized destination vector (in H<sub>2</sub>O)
*Purified, linearized destination vector (in H<sub>2</sub>O)
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#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube
#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube
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#Add 1 &mu;L ligation buffer to the tube.  <br>Vortex buffer before pipetting to ensure that it is well-mixed. <br>Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.  It is recommended that you aliquot the Ligation Buffer into smaller quantities.
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#Add ligation buffer.  <br>Vortex buffer before pipetting to ensure that it is well-mixed. <br>Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.  It is recommended that you aliquot the Ligation Buffer into smaller quantities.
#Add prefix part, suffix part and destination vector to the tube.
#Add prefix part, suffix part and destination vector to the tube.
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#Add T4 DNA ligase. <br>Vortex ligase before pipetting to ensure that it is well-mixed.  <br>Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip.  To ensure you add the correct amount, just touch your tip to the surface of the liquid when pipetting.
+
#Add T4 DNA Ligase. <br>Vortex ligase before pipetting to ensure that it is well-mixed.  <br>Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip.  To ensure you add the correct amount, just touch your tip to the surface of the liquid when pipetting.
#Incubate 20 minutes on the benchtop at room-temperature.
#Incubate 20 minutes on the benchtop at room-temperature.
#Place on ice until transformation.
#Place on ice until transformation.
[[Category:DNA]] [[Category:In vitro]] [[Category:Protocol]]
[[Category:DNA]] [[Category:In vitro]] [[Category:Protocol]]

Revision as of 22:36, 17 March 2008

Materials

  • T4 DNA Ligase from NEB
  • 10X T4 DNA Ligase buffer
  • Deionized, sterile H2O
  • Purified, linearized destination vector (in H2O)
  • Purified, linearized prefix part (in H2O)
  • Purified, linearized suffix part (in H2O)

Ligation Mix

  • 2-4 μL each purified, linearized DNA
  • 1 μL 10X Ligase Buffer
  • 200 units T4 DNA Ligase

deionized H2O to 10μL

Procedure

  1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
  2. Add ligation buffer.
    Vortex buffer before pipetting to ensure that it is well-mixed.
    Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation. It is recommended that you aliquot the Ligation Buffer into smaller quantities.
  3. Add prefix part, suffix part and destination vector to the tube.
  4. Add T4 DNA Ligase.
    Vortex ligase before pipetting to ensure that it is well-mixed.
    Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add the correct amount, just touch your tip to the surface of the liquid when pipetting.
  5. Incubate 20 minutes on the benchtop at room-temperature.
  6. Place on ice until transformation.
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