Ligation Standard Protocol

Ligations are used to combine DNA fragments.

Protocol

1. Generally, use 20 μL for total ligation volume

2. Use 10% ligase

• ALWAYS add the ligase last to the ligation mixture!!!

3. Add the ligase buffer

• Buffers are 20X, so use 2 μL for each 10 μL total volume

4. Usually have between 2 and 6 μL of DNA in ligation mixture

• This can be altered depending on the results you desire. For example, if you have an insert you wish to add to a vector, sometimes you may want to have up to 3x as much insert as vector in your ligation mixture.

5. Balance of ligation mixture should be deionized water

6. Allow ligation to set in 4 oC fridge or on desktop overnight

• Fridge is preferred

Following these guidelines, your ligation mixtures should look similar to these:

EXAMPLE LIGATION 1:
2 μL DNA Vector
6 μL DNA Insert
4 μL Ligation Buffer
2 μL Ligase
6 μL dd water