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Ligation Protocol for 3A Assembly Method

• The following protocol is provided by bioworks:

1. Add 11µl of denionized water to 200µl PCR tube
2. Add 2µl of each of the digests to the tube
3. Add 2µl of 10X T4 DNA Ligase Reaction Buffer to tube
4. Add 1µl of the T4 DNA Ligase to the tube
5. Mix solution well
6. Incubate the reaction mix at room temperature for 10 minutes
7. Incubate the reaction mix at 80C for 20 minutes to inactivate enzymes
8. Store ligation mix at -20C