Assembly of TALENs with Golden Gate kit
Step 2: putting the blocks together
Mix the following:
- 150 ng of each partial RVD array
- 150 ng of the final RVD (pLR-HD, pLR-NG, pLR-NI, pLR-NN, or pLR-NK)
- 75 ng of destination vector (linearized with Esp3I and gel purified)
- 1 µl Esp3I
- 1 µl T4 ligase or Quick ligase
- 2 µl of T4 ligase buffer
- H2O to 20 µl
Run 2-10 digestion/ligation cycles:
- 37°C for 5 min.
- 16°C for 10 min.
- Goto 1
- 37°C for 15 min.
- 80°C for 5 min.
Transform into E. coli