Kai Yuet/Protocols:DNA Ligation

DNA Ligation (KPY)

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

[math]\displaystyle{ \rm{Insert\ Mass\ (ng)} = 2.5\times\left[\frac{\rm{Insert\ Length\ (bp)}}{\rm{Vector\ Length\ (bp)}}\right]\times \rm{Vector\ Mass\ (ng)} }[/math]

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.

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