Lidstrom:Ligation

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From iGem UW 2011:

  • 7uL insert
  • 1uL vector
  • 1uL T4 ligase buffer
  • 1uL T4 ligase
  • Incubate at 16oC overnight, or room temperature for 30 minutes to 1 hour.

To vary vector:insert ratio?

  • You can do this, and thorough people do. Try doing it fast and simple (as iGem does above) before trying laborious/precise calculations & ratios.

To use controls?

  • You can do this, and thorough people do. The most common controls to use are VCL (vector, cut + ligase) and VCNL (vector, cut, no ligase used). These help you assess the frequency of the plasmid backbone's recircularization in the presence and absence of ligase. If you see a lot of colonies in the VCL plate, you should screen more of your colonies because more of them will be false positives.
  • It often doesn't take longer to include these controls, so if you have enough DNA you can do it. But it does require additional antibiotic plates which require labor to make!