DNA Ligation

Gregory W. Broussard

Revised: 07/13/11

(Please use, change and disseminate freely!)

• 15 µl ligation final volume
• Vector mass should be 100 ng
• For vector and insert fragments of about the same size use 1:1 molar ratio
• If insert is much smaller (<1000bp) use 1:3 (vector:insert) ratio
• For blunt end ligations use a 1:5 (vector:insert) ratio

1. Use the following equation to determine the volumes of insert and vector:

bp insert                              bp vector

———–            =                ———–

X ng insert                        100 ng vector

• “X” equals the ng of insert for a 1:1 ratio
• for a 1:3 ratio multiply X times 3
• with this information and previous measurements of concentration, determine volumes of each to add to the ligation mix.

2. Set up ligation mix:

(total vol. 15 µl)

? µl diH2O

? µl vector (100 ng)

? µl insert (see formula)

1.5 µl ligase buffer

1.5 µl ATP (0.75 µl for blunt ends)

1 µl ligase

• If you are using the Fast-Link DNA ligase kit, incubate at RT for 1hour
  • Manufacturer recommends shorter time, but longer is better here
  • If one enzyme has a blunt end cut, you MUST use the amounts of ATP for blunt ligations (too much ATP can inhibit the ligation.)

3. Transform into your favorite bug!

4. You can store the ligation at 4 or -20 ºC.