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Procedure:

1. Run PCR reaction on all the parts to add golden primers onto the parts (the BsaI restriction sites are added in this step)

1. When setting the annealing temperatures, only half of the primer must anneal. The part that anneals is the part that is unique to each primer. Generally the lower case letters outputted in the benchling pdf from the tool

2. If the part you are inserting is inside a plasmid that is same antibiotic resistance as your final destination vector, you should gel extract the linear part with after the pcr reaction

3. PCR purify all pcr products

4. Set Up Golden Reaction on ice:

1. 75-100 ng of Destination Vector

2. 2:1 Molar Ratio (insert:vector)

3. 2 uL of NEB Golden Gate Buffer (10X)

4. 1 uL NEB Golden Gate Assembly Mix

5. Up to 20 uL of MilliQ Water

1. Add water first, then Buffer, DNA/Vector, and lastly Assembly Mix

5. Incubate reaction in thermocycler:

1. For 1-4 inserts: 37C 1hr → 55C 5min

2. For 5-10 inserts: (37C 1min → 16C 1min) x 30 → 55C 5min

3. For 5-10 inserts: (37C 5min → 16C 5min) x 30 → 55C 5min

6. Transform 2-4 uL of reaction product