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Old Golden Gate Assembly Protocols

This page contains a collection of old protocols for Golden Gate Assembly. All these protocols should still work - they just aren't compatible with the use of the new Golden Gate Assembly Kits that we have swapped to!

Assembly reaction

Part Plasmid Assembly: First Stage and Second Stage Assembly:

• 10 fmol of each part plasmid = 650 * insert length * 10x10^-6 = X ng
• 2 μL of 10× T4 DNA ligase buffer (Promega)
• 1 μL of BsmBI or BsaI-HFv2
• 1 μL of T4 DNA ligase (Promega)
• x μL water up to 20 μL total.

For easy part volume calculation: BroadHostRange_Reaction_Calculator.xlsx

** Promega T4 Ligase buffer has been shown to offer the highest activity for BsaI enzyme. Other T4 ligase enzymes will work, but Promega enzyme with Promega buffer is highest efficiency.

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Mix samples well by pipetting, then run the reaction on the thermocycler under appropriate conditions:

BsmBI

Step	Temperature	Time
1	42°C	1.5 min
2	16°C	3 min
Cycles 1-2:	Repeat 25x
3	50°C	5 min
4	80°C	10 min

BsaI

Step	Temperature	Time
1	37°C	1.5 min
2	16°C	3 min
Cycles 1-2:	Repeat 25x
3	37°C	5 min
4	60°C	10 min

• Transform 2 μL assembly reaction and plate recovery on LB + Selective Antibiotic

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Tips from New England Biolabs on ways to change the reaction conditions for difficult assemblies involving many parts:

• Different reaction times based on the number of inserts (link)
• Better fidelity for assembling many inserts using a constant reaction temperature: (link)

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• BroadHostRange_Reaction_Calculator.xlsx: BroadHostRange_Reaction_Calculator.xlsx