Kai Yuet/Protocols:DNA Ligation
DNA Ligation (KPY)
Following Gel Extraction of 1 μg Enzymatic Digestion Using Qiagen's QIAquick Gel Extraction Kit (Elution Volume 30μL of TE)
- Purified, Linearized Vector (in TE)
- Purified, Linearized Insert (in TE)
- 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
- Add appropriate amount of ddH2O to 1mL microcentrifuge tube.
- Add 1 μL of 10X T4 DNA Ligase Buffer to the tube.
- Add appropriate amount of insert to the tube.
- Add 2 μL of vector to the tube.
- Add 0.5 μL of T4 DNA Ligase to the tube.
- Incubate at 25°C for 20 minutes.
- Place on ice until transformation.
- 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.
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