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Assembly of TALENs with Golden Gate kit

Step 2: putting the blocks together

Mix the following:

  1. 150 ng of each partial RVD array
  2. 150 ng of the final RVD (pLR-HD, pLR-NG, pLR-NI, pLR-NN, or pLR-NK)
  3. 75 ng of destination vector (linearized with Esp3I and gel purified)
  4. 1 µl Esp3I
  5. 1 µl T4 ligase or Quick ligase
  6. 2 µl of T4 ligase buffer
  7. H2O to 20 µl

Run 2-10 digestion/ligation cycles:

  1. 37°C for 5 min.
  2. 16°C for 10 min.
  3. Goto 1
  4. 37°C for 15 min.
  5. 80°C for 5 min.

Transform into E. coli