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Digestion Protocol for 3A Assembly Method

• The following was protocol provided by bioworks:

1. Determine upstream part, downstream part and destination plasmid
2. Label three PCR tubes as U, D, P respectively
3. To each tube add 500ng of plasmid to be digested
4. Add necessary amount of water so final volume is 42.5µl
5. Add 5µl of NEBuffer 2 to each tube
6. Add 0.5µl of BSA to each tube
7. Add 1µl of first appropriate enzyme
8. Add 1µl of second appropriate enzyme
9. Ensure that digest is well mixed
10. Incubate the digests at 37C for 15 minutes in thermocycler
11. Incubate the digests at 80C for 20 minutes to deactivate the restriction enzymes
12. Store digest at -20C

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