From 2014.igem.org
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 ATP |
1.00 | 1 T4 DNA Ligase |
- | 20-100 ng linear vector DNA |
- | 100-500 ng insert DNA |
ad 20 µl H2O | | Cycler Program | Step | Temperature [°C] | Time [min] | Cycle no. | 1. | 22 | 60.0 | 1 | 2. | 80 | 10.0 | 1 |
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