Kai Yuet/Protocols:DNA Ligation

From OpenWetWare
Jump to: navigation, search

DNA Ligation (KPY)

Following Gel Extraction of 1 μg Enzymatic Digestion Using Qiagen's QIAquick Gel Extraction Kit (Elution Volume 30μL of TE)

Materials

  • Purified, Linearized Vector (in TE)
  • Purified, Linearized Insert (in TE)
  • ddH2O
  • 10X T4 DNA Ligase Buffer
  • T4 DNA Ligase

10 μL Ligation Mixture

  • 2 μL Vector (approximately 40 ng)
  • x μL Insert (2.5-fold molar excess)
  • 1 μL 10X T4 DNA Ligase Buffer
  • (6.5 - x) μL ddH₂O
  • 0.5 μL T4 DNA Ligase

Calculating Insert Amount

Procedures

  1. Add appropriate amount of ddH2O to 1mL microcentrifuge tube.
  2. Add 1 μL of 10X T4 DNA Ligase Buffer to the tube.
  3. Add appropriate amount of insert to the tube.
  4. Add 2 μL of vector to the tube.
  5. Add 0.5 μL of T4 DNA Ligase to the tube.
  6. Incubate at 25°C for 20 minutes.
  7. Place on ice until transformation.

Notes

  • T4 DNA Ligase Buffer (aliquot) should be completely dissolved and vortexed before use.
  • Mid-October ligation/transformation failures were resolved with a change of loading dye following enzymatic digestion of DNA.

Back to Kai's Notebook.