Barrick Lab :: Old_Golden_Gate

[[GoldenGateAssemblyProtocolsMainPage][Back to Golden Gate Protocols]]

---+ 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:
<div style="padding: 1%">
   * 10 fmol pYTK-001 plasmid = *17.7ng*  [try to keep volume to 1-2&mu;L]
   * 20 fmol of insert DNA = 650 * insert length * 20x10^-6 = *X ng* [try to keep volume to less than 10 &mu;L]
   * 2 &mu;L of 10× T4 DNA ligase buffer (Promega)
   * 1 &mu;L of BsmBI
   * 1 &mu;L of T4 DNA ligase
   * x &mu;L water up to *20 &mu;L total*
</div>
First Stage and Second Stage Assembly:

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

For easy part volume calculation: [[%ATTACHURL%/BroadHostRange_Reaction_Calculator.xlsx][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 &mu;L 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 ([[https://www.neb.com/protocols/2015/03/04/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1600][link]])
   * Better fidelity for assembling many inserts using a constant reaction temperature: ([[https://www.biorxiv.org/content/10.1101/2020.12.22.424019v1][link]])

[[GoldenGateAssemblyProtocolsMainPage][Back to Golden Gate Protocols]]
   * [[%ATTACHURL%/BroadHostRange_Reaction_Calculator.xlsx][BroadHostRange_Reaction_Calculator.xlsx]]: BroadHostRange_Reaction_Calculator.xlsx

Attachments

Topic attachments
I	Attachment	History	Action	Size	Date	Who	Comment
xlsx	BroadHostRange_Reaction_Calculator.xlsx	r1	manage	17.7 K	2021-11-03 - 21:02	KateElston

Barrick Lab > BroadHostRangeToolkit > ProtocolsBTKAssembleSingleTUPlasmid > Old_Golden_Gate_Assembly

Contributors to this topic

KateElston

Topic revision: r1 - 2021-11-03 - 21:05:10 - Main.KateElston