Barrick Lab :: ProtocolsBTKAssembleSingleTUPlasmid

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

---+ First-Stage Plasmid (Transcriptional Unit) Assembly

Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present ([[http://barricklab.org/twiki/bin/view/Lab/ProtocolsBTKDesignANewPart Described here]]).

Protocol source: NEB ([[https://www.neb.com/protocols/2018/10/02/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1601]])

---+++ Assembly reaction
[[Old_Golden_Gate_Assembly][Access the old non-kit golden gate assembly protocols here]]

Total volume will be 20 &mu;L; you will need 50 fmol of each part plasmid for this assembly. Typically the pYTK095 Type 6-8 plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place.

   * 50 fmol of each part plasmid = 650 * insert length * 50x10^-6 = *X ng*
   * 2 &mu;L of NEB 10× T4 DNA ligase buffer
   * 1 &mu;L of NEB Golden Gate Enzyme Mix BsaI-HFv2 (**DO NOT* use BsaI or BsaI-HF)
   * x &mu;L water up to 20 &mu;L total.

For easy part volume calculation: [[%ATTACHURL%/GGA_Kit_Reaction_Calculation_Spreadsheet.xlsx][GGA_Kit_Reaction_Calculation_Spreadsheet.xlsx]]

*BsaI and BsaI-HF don't have high fidelity and/or activity under the Golden Gate buffer/temperature conditions.

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

|  *Step*  |  *Temperature*  |  *Time*  |
|  1  |  37°C  |  1 min  |
|  2  |  16°C  |  1 min  |
| Cycles 1-2: | Repeat 30x | |
|  3  |  60°C  |  5 min  |

 Transform 2 &mu;L of the assembly reaction and plate on an appropriate antibiotic (Amp/Carb if using pYTK095).

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]])

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Attachments

Topic attachments
I	Attachment	History	Action	Size	Date	Who	Comment
xlsx	GGA_Kit_Reaction_Calculation_Spreadsheet.xlsx	r2 r1	manage	12.1 K	2021-11-03 - 20:28	KateElston

Barrick Lab > BroadHostRangeToolkit > ProtocolsBTKAssembleSingleTUPlasmid

Contributors to this topic

KateElston, SeanLeonard, DennisMishler, JeffreyBarrick, VictorLi, PatrickLariviere

Topic revision: r18 - 2021-11-03 - 20:57:10 - Main.KateElston