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)====

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

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.