Ligations using Promega T4 DNA Ligase

** See Product Info Sheet for more information

Use a 1:1, 1:3, or 3:1 molar ratio of vector:insert DNA when cloning a fragment into a plasmid vector.  These ratios will vary with other types of vectors, for example, cDNA and genomic cloning vectors.  The following example illustrates the conversion of molar ratios to mass ratios for a 3.0kb plasmid and a 0.5kb insert DNA fragment.

ng of vector x kb size of insert  x molar ratio of insert/vector = ng of insert

            kb size of vector

Example: 

How much 0.5kb insert DNA should be added to a ligation in which 100ng of 3kb vector will be used?  The desired vector:insert ratio will be 1:3.

100ng vector x 05.kb insert  x  3/1 = 50ng insert

            3kb vector

The following ligation reaction of a 3kb vector and a 0.5kb insert DNA uses a 1:1 vector: insert ratio.  Typical ligation reactions use 100-200ng of vector DNA.

  1. Assemble the following reaction in a sterile microcentrifuge tube:

Vector DNA                                       100ng

Insert DNA                                          17ng

Ligase 10X buffer                                1ul

T4 DNA Ligase (Weiss units)        0.1-1u

Nuclease free water                        to 10ul

  1. Incubate reaction at:

Room temperature for 3 hours, or

4C overnight, or

15C for 4-18 hours.