Subcloning Tips - very important heating step
|Date Added: Wed Sep 25 2002|
|Date Modified: Wed Sep 25 2002|
|Abstract: Put your insert and vector in an Eppendorff tube and then heat it for 5 minutes, then add ligation buffer and ligase|
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.
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.