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From 2010.igem.org

Module I, group 2(b)

Digest the four fragments with restriction enzyme separately except the SalI and EcoRI at 37℃ for more than 3h. Ligate the four fragments at 25℃ for an hour.

Ligation system:

 H2O	        22.5μl
 10×buffer	5μl
 fragments	5μl each
 T4 ligase	2.5μl
 Total	        50μl

Run a gel.

Result

There isn’t any ligation product exists… Even though it exists, the concentration is not high enough to be detected. We’ll improve the ligation protocol!