• Thaw all reagents on ice.
  • Assemble reaction mix into 10 µL volume in a microfuge tube. Reaction may be scaled up to 20 µL if DNA concentrations are low.
  • Add reagents in following order: water, buffer, insert, vector, T4 ligase.
  • Gently mix by stirring gently with pipette tip.
  • Typical Incubation time  and temperature is 15°C for at least 4 hours. Incubation at 4°C for overnight is commonly used.
  • Prepare a control that excludes the insert. This allows for detection of re-annealing due to  incomplete vector digestion (If vector is not completely digested  the colonies generated by the vector reaction will greatly outnumber that of the vector + insert reaction).
  • Ligation Reaction can be inactivate d by incubation at 65°C for 20 minutes. This step is optional.
  • Proceed directly to transformation reaction.
Component Final concentration/amount
water
to 10 µL
Ligase buffer (with ATP)
1X
Vector
25 ng
Insert
75 ng
T4 DNA ligase
0.1 to 1 Weiss  unit

REQUEST A QUOTE

feedback

Do you like the current new website?

Hate

Dislike

Neutral

Like

Love

*