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Revision as of 11:21, 14 October 2014

Protocols / Ligation

After restriction enzyme cleavage, compatible ends of two fragments can hybridize, but are not stable due to their missing phosphodiester bonds. To fix those nicks in the double-stranded DNA, a ligase from T4 phages (Thermo Scientific) was utilized. The ligation reaction mix is shown in the following table.

Table 1: Reaction mixes and temperature programs for the ligation.

Volume [μl] Compounds of the digest 2.00 10 x T4 DNA Ligase buffer 2.00 10 mM µl-1 ATP 1.00 1 U T4 DNA Ligase - 20-100 ng linear vector DNA - 100-500 ng insert DNA ad 20 µl H2O

Cylcer Program Step Temperature [°C] Time [min] Cycle no. 1. 22 60.0 1 2. 80 10.0 1