Protocol Online: Subcloning Tips - very important heating step

Subcloning Tips - very important heating step

Source: Schimmelpenninck
Abstract: Put your insert and vector in an Eppendorff tube and then heat it for 5 minutes, then add ligation buffer and ligase
Date Added: 2002-09-25
Date Modified: 2002-09-25

After isolating your digested fragments from the gel put digested vector and digested insert TOGETHER in an Eppendorff tube, then put this for about 5 minutes at 65 degrees celsius, then cool it and spin it down, then add your ligation buffer and ligase. The 65 degrees celsius step is VERY important.

Short explanation:
your insert is EcoRI/SalI
and vector is EcoRI/SalI
The heating step will break all the UNWANTED AND TOTALLY USELESS insert-insert and vector-vector bindings. After the heating step MANY good vector insert bindings will occur and your plates will be full of good transformants.