A modified LIC protocol was developed in our laboratory
for directional cloning of PCR-amplified DNA directly as
in-frame fusions to SUMOpro or SUMOstar.
The protocol is illustrated below.

• The gene of interest (GOI) is amplified using a pair
of gene specific primers that include 20 base extensions
complementary of our linearized vector.
• Our modified PCR protocol creates sufficient PCR products
with 5’ overhangs for LIC.
• Vector and insert DNA are mixed, incubated for a short time,
and transformed into competent E. coli cells.

The main advantage of the protocol is that it completely
avoids restriction digestion and ligation of the insert DNA.
This is especially useful for large genes where internal
restriction sites can interfere with conventional cloning.
It can also be easily adapted for high throughput cloning.

The strategy for ligation independent cloning (LIC).