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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:
  • 10 fmol pYTK-001 plasmid = 17.7ng [try to keep volume to 1-2μL]
  • 20 fmol of insert DNA = 650 * insert length * 20x10^-6 = X ng [try to keep volume to less than 10 μL]
  • 2 μL of 10 T4 DNA ligase buffer (Promega)
  • 1 μL of BsmBI
  • 1 μL of T4 DNA ligase
  • x μL water up to 20 μL total
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.


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

BsmBI

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

BsaI

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

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


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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Microsoft Excel Spreadsheetxlsx BroadHostRange_Reaction_Calculator.xlsx r1 manage 17.7 K 2021-11-03 - 21:02 KateElston  
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